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International Symposium on Problems of Listeriosis

Ficha Técnica
LIVRO DE ACTAS DO CONGRESSO: ISOPOL XVII
Intrenational Symposium on Problems of Listeriosis
Editor: Universidade Católica Portuguesa – Escola Superior de Biotecnologia
Coordenação e Revisão: Paula Teixeira
Design e Composição Gráfica: Kai Sprecher / Lynn Salt
Impressão: Orgal Impressores
Depósito Legal:
Tiragem: 500 exemplares

WELCOME TO ISOPOL XVII
On behalf of the Organising Committee, I am pleased to welcome you to
ISOPOL XVII (International Symposium On Problems Of Listeriosis) which
this year is organized in Porto by the Universidade Católica Portuguesa –
Escola Superior de Biotecnologia.
The ISOPOL meetings provide a unique opportunity for interdisciplinary
discussions concerning various aspects of listeriosis including, but not being
limited to, food safety and clinical aspects. Since the first edition of this
symposium in Giessen, Germany (1957), much has been discovered and
many more questions have been posed. For this reason the underlying motives
for bringing together the evolving ISOPOL community are still very
strong in 2010.
More than 350 delegates from the clinical, veterinary, and public health
areas as well as the food industry, will be present, representing ca. 45 countries.
This diversity will certainly provide for a very stimulating and plural
atmosphere which is sure to promote rich discussions and exchanges of
ideas.
On this occasion, we would like to thank to the members of the Scientific
Committee for their time and effort in maintaining the high scientific level
of ISOPOL XVII.
We are also thankful to our sponsors, which, in many different ways, have
greatly contributed to the success of the organization of this event.
For the first time ever, ISOPOL will take place in Portugal, namely in Porto
– the country’s second city and the capital of the north.
It is easy to see why Porto was designated a World Heritage Site by UN-
ESCO in 1996. It is a living city, full of monuments and signs of its rich history.
It is best met in person; take the opportunity to stay awhile, to meet
the people, to enjoy the charms of this city of contrasts – you may find the
need to return again to enjoy it all!
Paula Cristina Maia Teixeira

TABLE OF CONTENTS
KEY NOTE SPEECH
Listeria monocytogenes: a multifaceted model 21
Cossart, P.
AREA //A // Biology of Listeria monocytogenes
PLENARY LECTURES
A / PL/ 01 The pangenome of Listera spp. 22
Chakraborty, T.
A / PL/ 02 Ecology of L. monocytogenes and Listeria spp. in natural 22
and food associated environments
Wiedmann, M.
ORAL PRESENTATIONS
A / O/ 01 The cold shock associated proteins (Csps) promote tolerance 33
of different environmental stresses and host cell invasion
of Listeria monocytogenes
Tasara, T., Klumpp, J., Loessner, M. J. and Stephan, R.
A / O/ 02 Different levels of flagellin detected during growth of 33
Listeria monocytogenes strains at low temperature
Cabrita, P., Batista, S., Moes, S., Jenö, P., Trigo, M. J., Boavida Ferreira, R. and Brito, L.
A / O/ 03 Phenotypic and corresponding transcriptomic responses of 34
Listeria monocytogenes strains in the presence of
unprotonated organic acids
Lee Chang, K. J., Pinfold, T., Koshy, A. and Bowman, J. P.
A / O/ 04 Internalin profiling, multilocus sequence typing and 34
virulence assesments suggest evolutionary history of the
Listeria monocytogenes-Listeria innocua clade
Chen, J. and Fang, W.
A / O/ 05 The SOS response of Listeria monocytogenes is involved 35
in stress resistance, mutagenesis, and biofilm formation
van der Veen, S. and Abee, T.
A / O/ 06 Clonal diversity of Listeria monocytogenes, a worldwide perspective 35
Chenal-Francisque, V., Lopez, J., Cantinelli, T., Caro, V., Tran, C., Leclerq, A.,
Lecuit, M. and Brisse, S.
A / O/ 07 Life without a cell wall: Listeria monocytogenes L-form cells 36
feature a unique mode of division
Briers, Y., Dell’Era, S., Schuppler, M. and Loessner, M. J.
A / O/ 08 Pangenomic analysis of Listeria monocytogenes 36
Deng, X., Phillippy, A. M., Li, Z., Salzberg, S. L., Tortorello, M. L. and Zhang, W.
A / O/ 09 Evidence for an antiporter-independent glutamate decarboxylase 37
(GAD) system in Listeria monocytogenes: Influence of growth media
on GAD system activity
Karatzas, K.-A., Brennan, O., Heavin, S. and O’Byrne, C. P.
A / O/ 10 Role of Listeria monocytogenes tyrosine phosphatases 37
in conferring listeriophage resistance
Paz, R.-N., Eugster, M. R., Zeiman, E., Loessner, M. J. and Calendar, R.
A / O/ 11 Thiolomics – the thiol: disulfide redox metabolism of Listeria monocytogenes 38
Ondrusch, N., Gopal, S., Fuss, A., Hagen, N., Stoll, R., Aharonowitz, Y. and Kreft, J.

A / O/ 12 RNA-structures acting at a distance 38
Johansson, J.
A / O/ 13 Deep RNA sequencing of Listeria monocytogenes reveals overlapping 39
and extensive stationary phase and sigma B-dependent transcriptomes,
including multiple highly transcribed noncoding RNAs
Oliver, H. F., Orsi, R. H., Ponnala, L., Keich, U., Wang, W., Sun, Q., Cartinhour, S.,
Filiatrault, M. J., Wiedmann, M. and Boor, K. J.
POSTER PRESENTATIONS
A / P/ 00 Compartmentalization of IFN-gamma and IL-6 receptors signalling 62
in Listeria monocytogenes phagosomes: immune vesicles
Ramos-Vivas, J., Carrasco-Marin, E., Madrazo-Toca, F., Fernandez-Prieto, L.,
Rodriguez-Del Rio, E., Carranza-Cereceda, C. and Alvarez-Dominguez, C.
A / P/ 01 Antimicrobial susceptibility among Listeria monocytogenes isolates 63
from non human sources in France over a ten year period
Granier, S. A., Moubarek, C., Colaneri, C., Roussel, S., Courvalin, P. and Brisabois, A.
A / P/ 02 Five homologous small RNAs are involved in the response 63
of Listeria monocytogenes to cell wall acting antibiotics
Kiil Nielsen, P. and Kallipolitis, B.
A / P/ 03 Phenotypic analysis of selected listerial secretion mutants 64
Halbedel, S., Galander, S. and Flieger, A.
A / P/ 04 Distribution of serotypes and pulsotypes of L. monocytogenes 64
in pig farms (France 2008)
Boscher, E.
A / P/ 05 Comparative phylogenomics of Listeria monocytogenes reveals 65
an adaptation profile
Silveira Nalério, E., Padilha Silva, W., Stabler, R. and Wren, B. W.
A / P/ 06 Serotyping and PFGE patterns of Listeria monocytogenes isolated 65
from poultry meat
Vasantrao Kurkure, N., Kalorey, D. R., Rodrigues, J., Gunjal, P.1 and Barbuddhe, S. B.
A / P/ 07 Genotypic characterization of Listeria monocytogenes isolated 66
from fresh leafy vegetables
Warke, S., Kalorey, D. R., Umap, S., Sonegaonkar, A., Patil, V., Kurkure, N V. and Barbuddhe, S. B.
A / P/ 08 Comprehensive appraisal of the exoproteome of Listeria monocytogenes 66
by genomic and proteomic analyses
Desvaux, M., Dumas, E., Chafsey, I., Chambon, C. and Hébraud, M.
A / P/ 09 Investigating differences in lineages of Listeria monocytogenes 67
using comparative genomics
McIlwham, S., Farber, J. and Pagotto, F.
A / P/ 10 Autolysis in Listeria monocytogenes – a proteomic approach 67
Pinto, E., Marques, N., Andrew, P. W. and Faleiro, M. L.
A / P/ 11 Role of flhA, cheR and motA in growth of Listeria monocytogenes 68
at low temperature
Mattila, M., Lindström, M., Somervuo, P. and Korkeala, H.
A / P/ 12 The effect of acetic acid (at pH 5.5) or benzoic acid (at neutral pH) on lipid 68
composition and fluidity of Listeria monocytogenes membrane
Ioannis, D., Anita, B., Eleni, S. and Mastronicolis, S.
A / P/ 13 Elucidation of the responses to weak acids in the human pathogen 69
Listeria monocytogenes using gene microarrays
O’Byrne, C., Heavin, S. and Morrissey, J.
A / P/ 14 The immunogenic surface protein IspC acts as an N-Acetylglucosaminidase 69
in Listeria monocytogenes serotype 4b
Ronholm, J.
A / P/ 15 Listeria monocytogenes EGD chitinolytic activity is regulated 70
by carbohydrates but also by the virulence regulatory gene, PrfA
Halberg Larsen, M., Leisner, J. J. and Ingmer, H.
A / P/ 16 Infectious dose curves for guinea pigs challenged with a Listeria 70
monocytogenes epidemic clone strain and a strain carrying a
naturally-occurring virulence-attenuating mutation in inlA show a
significant shift in median infectious dose
Nightingale, K., Van Stelten, A., Simpson, J. M., Chen, Y., Scott, V. N., Ross, W. H.,
Whiting, R. C. and Wiedmann, M.
A / P/ 17 MudPIT based proteomic analysis of alkaline adapted, environmentally 71
persistent Listeria monocytogenes strains
Nilsson, R. E., Ross, T. and Bowman, J. P.
A / P/ 18 RpoN, the alternative sigma factor, is associated with the growth phase 71
transition and pathogenesis in Listeria monocytogenes
Okada, Y., Suzuki, H., Monden, S., Igimi, S. and Okada, N.

A / P/ 19 Cellular lipid fatty acid pattern differences between reference 72
and ice-cream isolate of Listeria monocytogenes as response to cold stress
Anita, B., Ioannis, D. and Mastronicolis, S.
A / P/ 20 Role of the dihydroxyacetone metabolism in the resistance 72
of Listeria innocua to pediocin
Milohanic, E.
A / P/ 21 Glucose transport system in Listeria monocytogenes 73
and their impact on virulence gene expression
Moussan Ake, F.
A / P/ 22 Antimicrobial susceptibilities of Listeria monocytogenes isolated in Japan 73
Monden, S., Okutani, A., Suzuki, H., Asakura, H., Nakama, A., Igimi, S., Okada, Y. and Maruyama, T.
A / P/ 23 Influence of sub-lethal concentrations of disinfectants 74
on Listeria monocytogenes adhesion and invasion in Caco-2 cells
Gaedt Kastbjerg, V., Halberg Larsen, M., Ingmer, H. and Gram, L.
A / P/ 24 The SOS response in Listeria monocytogenes – a stress survival mechanism 74
Kiil Nielsen, P., Zahle Andersen, A. and Haahr Kallipolitis, B.
A / P/ 25 Acid shock triggers heavy metal detoxification in Listeria monocytogenes 75
Müller, S., Neuhaus, K. and Scherer, S.
A / P/ 26 Listeria monocytogenes mutants defective in growth at 5 °C 75
and in high salt environment
Burall, L., Laksanalamai, P. and Datta, A.
A / P/ 27 Revival of 5,000 Listeria strains from Seeliger’s historical collection 76
with a semi-automated microbiological pipeline
Haase, J., Hof, H. and Achtman, M.
A / P/ 28 Two point mutations are responsible for the lack of glycosidic substition 76
in cell wall teichoic acids in Listeria monocytogenes serovar “7”
Eugster, M. R., Huwiler, S., Morax, L. and Loessner, M. J.
A / P/ 29 High-throughput genome sequencing of two Listeria monocytogenes 77
clinical isolates during a large foodborne outbreak
Gilmour, M., Graham, M., Van Domselaar, G., Tyler, S., Kent, H., Trout-Yakel, K.,
Larios, O., Allen, V., Lee, B., Nadon, C. and Kearney, A.
A / P/ 30 Expression of antimicrobial activity in food 77
and clinical Listeria monocytogenes isolates
Barbosa, J., Ferreira, V., Borges, S., Azevedo, I., Magalhães, R., Santos, I, Almeida, G. and Teixeira, P.
A / P/ 31 The Listeria monocytogenes sigma B and sigma H regulons overlap, 78
but only sigma B appears to be important for survival of acid,
alkaline and oxidative stress
Chaturongakul, S., Raengpradub, S., Wiedmann, M. and Boor, K. J.
A / P/ 32 Virulence gene expression in Listeria monocytogenes strains isolated 78
from different sources
Alessandria, V., Rantsiou, K. and Cocolin, L.
A / P/ 33 Differentiation of Listeria monocytogenes, Listeria innocua 79
and Listeria marthii, a novel Listeria species isolated
from the natural environment, Finger Lakes National Forest
Graves, L. M., Helsel, L. O., Steigerwalt, A. G., Morey, R. E., Daneshvar, M. I., Roof, S. E.,
Orsi, R. H., Fortes, E. D., Milillo, S. R., den Bakker, H. C., Wiedmann, M.,
Swaminathan, B. and Sauders, B. D.
A / P/ 34 Differed roles of L,D-carboxypeptidases encoded by lmo0028 79
and lmo1638 genes
Yurov, D., Varfolomeev, A., Kaminskaya, A. and Ermolaeva, S.
A / P/ 35 Antimicrobial susceptibilities of Listeria monocytogenes isolated 80
from retail beef, pork and poultry in Japan
Ida, M., Shimojima, Y., Kaneko, S., Higuchi, Y., Nakama, A. and Kai, A.
A / P/ 36 Genetic basis of two low pathogenic L. monocytogenes strains 80
with apparent phospholipase C activity
Jiang, L., Bai, F., Chen, J., and Fang, W.
A / P/ 37 Molecular genotyping and antimicrobial resistance 81
of Listeria monocytogenes from foods and the environment
Parisi, A., Miccolupo, A., Fraccalvieri, R., Latorre, L., Normanno, G. and Santagada, G.
A / P/ 38 Virulence transcriptome analysis of Listeria monocytogenes 81
by application of microarrays in vitro and in situ
Rantsiou, K., Alessandria, V. and Cocolin, L.
A / P/ 39 Effects of growth conditions on surface properties of Listeria; 82
a proposed role for AI-2
Wong, H. T. L., Nwaiwu, O. and Rees, C. E. D.
A/P/40 Stress behaviour of a Listeria monocytogenes 568 Lmo1634 transposon mutant 82
Truelstrup Hansen, L., Holman, D. B. and Ells, T. C.

A/P/41 Investigation of the conditions that trigger the activation 83
of the alternative sigma factor σB in Listeria monocytogenes
Utratna, M., Shaw, I. and O’Byrne, C.
A/P/42 Characterization of Listeria monocytogenes 1/2a, 1/2b, 1/2c and 4b 83
by amplified fragment length polymorphism and evaluation
of their geographical distributions in Portugal
Maia, C. H., Goulão, M. M., Santos, M. I., Ferreira, M. A. S. S. and Pintado, C. M. B. S.
A/P/43 Global analysis of the Listeria monocytogenes surface proteins 84
of the LPXTG family
Botello-Morte, L., Calvo, E., Mariscotti, J., D’Orazio, V., García-del Portillo, F. and Pucciarelli, M. G.
A/P/44 Antimicrobial susceptibility of Listeria monocytogenes strains 84
derived from food and food-processing bakery plant
Eusébio, C., Carneiro, L., Santos, I., Magalhães, R., Almeida, G., Silva, J. and Teixeira, P.
A/P/45 A physiological study to purpose a new formal method 85
to obtain L. monocytogenes cells adapted to BAC
Saá Ibusquiza, P., Cabo, M. L., Herrera, J. J. R., Vázquez, D., Carrera, S. and Eiriz, E.
A/P/46 Characterization of a RNA-helicase in the human pathogen 85
Listeria monocytogenes
Netterling, S. and Johansson, J.
A/P/47 Listeria monocytogenes agr system: Quorum sensing, or maybe not? 162
Garmyn, D., Révelin, C. and Piveteau, P.
AREA //B // Listeria monocytogenes as a human and animal pathogen
PLENARY LECTURES
B / PL/ 03 How Listeria monocytogenes breaches host barriers 23
Lecuit, M.
B / PL/ 04 Secretion of a novel L. monocytogenes cyclic dinucleotide 23
into the cytosol of infected host cells activates an innate immune pathway
Portnoy, D. A.
B / PL/ 05 Diagnosis and clinical management of listeriosis in ruminants and camelids 24
Poulsen, K. P.
B / PL/ 06 Listeriolysin S – a second haemolysin with a role 24
in the virulence of Listeria monocytogenes
Hill, C.
ORAL PRESENTATIONS
B / O/ 14 Probiotics reduce Listeria monocytogenes-induced tissue invasion 40
and stillbirths in pregnant guinea pigs
Smith, M. A., Agyekum, K. and Williams, D.
B / O/ 15 Listeriolysin O favors Listeria monocytogenes growth 40
in co-culture with the ciliate Tetrahymena pyriformis
Ermolaeva, S. and Pushkareva, V.
B / O/ 16 Atopy is a risk factor for listeriosis 41
Kawamoto, K., Matsubara, S., Da Silva, M. and Makino, S.-I.
B / O/ 17 Cancer immunotherapy using novel Listeria monocytogenes-bacterial 41
vectors to target the vasculature of progressive tumors
Paterson, Y., Seavey, M. M., Maciag, P. C. and Sewell, D.
B / O/ 18 The intracellular carbon metabolism of Listeria monocytogenes 42
in comparison to that of other intracellular bacterial pathogens
replicating in mammalian host cells
Gotz, A., Eylert, E., Stoll, R., Eisenreich, W., and Goebel, W.
B / O/ 19 LIMP2 links late phagosomal trafficking with the onset of the innate 42
immune response to Listeria monocytogenes: a role in macrophage activation
Fernandez-Prieto, L., Carrasco-Marin, E., Madrazo-Toca, F., Rodriguez-Del Rio, E.,
Carranza-Cereceda, C. and Alvarez-Dominguez, C.
B / O/ 20 The tetraspanin CD81 is required for entry of Listeria monocytogenes 43
in mammalian cells
Tham, T. N., Gouin, E., Rubinstein, E., Boucheix, C., Cossart, P. and Pizarro-Cerdá, J.
B / O/ 21 Listeria innate immune evasion by peptidoglycan modification 43
Aubry, C.
B / O/ 22 Constitutive activation of the central virulence transcriptional regulator 44
PrfA enhances Listeria monocytogenes pathogenesis
but reduces bacterial fitness outside of the host
Freitag, N. E. and Bruno, J. C.
B / O/ 23 The lvfH gene of Listeria monocytogenes encodes a novel 44
virulence factor highly activated during infection
Carvalho, F., Camejo, A., Ferreira, P., Sousa, S. and Cabanes, D.

POSTER PRESENTATIONS
B / P/ 47 A Listeria monocytogenes strain is still virulent despite non-functional 86
major virulence genes: Optical Mapping shows a potential mechanism
Roche, S. M., Grépinet, O., Corde, Y., Teixeira, A. P., Kerouanton, A., Témoin, S.,
Mereghetti, L., Brisabois, A. and Velge, P.
B / P/ 48 Protein expression of lineage I, II, and III Listeria monocytogenes 86
strains in murine macrophages
Donaldson, J. R., Nanduri, B., Pittman, J. R., Burgess, S. C. and Lawrence, M. L.
B / P/ 49 Prevalence of L. monocytogenes in bovine mastitic milk samples: 87
Possible source of food borne infection
Kalorey, D. R., Warke, S., Kurkure, N. V. and Barbuddhe, S. B.
B / P/ 50 Agr-dependent peptide sensing in L. monocytogenes – Effects on 87
biofilm formation, virulence and global gene expression
Waidmann, M. S., Monk, I. R., Auchter, M., Preising, N. P., Hill, C. and Riedel, C. U.
B / P/ 51 agrD-deletion affects InlA- and InlB-regulation via 88
a temperature-dependent and -independent mechanism
Waidmann, M. S., Monk, I. R. and Riedel, C. U.
B / P/ 52 Bovine cranial nerve Schwann cells express E-cadherin, 88
a candidate key-player in the brainstem invasion
of Listeria monocytogenes in cattle
Madarame, H., Seuberlich, T., Vandevelde, M., Zurbriggen, A., and Oevermann, A.
B / P/ 53 Pathogenic potential of Listeria monocytogenes isolates 89
from New Zealand seafood premises: implications for control
Durante Cruz, C. and Fletcher, G.
B / P/ 54 Copper homeostasis and virulence in Listeria monocytogenes 89
David, C., Schuler, S., Glenn, S., Jen, C., Andrew, P. and Roberts, I. S.
B / P/ 55 Pattern of cytokine production during murine listeriosis 90
Dussurget, O.
B / P/ 56 Analysis of the post-translocation chaperone PrsA2 and its unique 90
role in facilitating Listeria monocytogenes pathogenesis
Alonzo, F. and Freitag, N.
B / P/ 57 CtaP is a multifunctional cysteine-transport associated protein 91
required for Listeria monocytogenes pathogenesis
Xayarath, B.
B / P/ 58 Enzymatic activity of the metalloprotease of Listeria is regulated by pH 91
Forster, B. M., Pavinski Bitar, A., Slepkov, E. R. and Marquis, H.
B / P/ 59 Identification of propeptide residues regulating the compartmentalization, 92
maturation, and activity of the broad-range phospholipase C
of Listeria monocytogenes
Slepkov, E. R., Pavinski Bitar, A. and Marquis, H.
B / P/ 60 A mouse model of fetoplacental Listeria monocytogenes 92
infection and abortion
Poulsen, K. P., Faith, N., Laura Knoll, L. and Czuprynski, C.
B / P/ 61 Listeria infection of the insect model system Galleria mellonella 93
Joyce, S. A. and Gahan, C. G.
B / P/ 62 Construction of a murinised Listeria monocytogenes H7858 (4b) 93
strain for improved murine infection
Cummins, J. and Gahan, C.
B / P/ 63 The role of a phosphoinositide phosphatase in the intracellular survival 94
of Listeria monocytogenes
Wang, J., Corbett, D. and Roberts, I. S.
B / P/ 64 Virulence gene expression in Listeria monocytogenes strains isolated 94
from different sources
Alessandria, V.
B / P/ 65 Targeted Signature-tagged mutagenesis for phenotype screening 95
of Listeria monocytogenes mutants
Henriques, A., Carvalho, F. and Cabanes, D.
B / P/ 66 Listeria monocytogenes cellular infection triggers 95
tyrosine-phosphorylation of Myosin IIA, a new protein
involved in invasion
Almeida, M. T., Cabanes, D. and Sousa, S.
B / P/ 67 Invasion profile of Listeria monocytogenes strains involved 96
in invasive and gastroenteritis listeriosis outbreaks
Laksanalamai, P., Sahu, S. and Datta, A.
B / P/ 68 Molecular characterization of the Vip-Gp96 interaction 96
Martins, M., Cabanes, D. and Sousa, S.
B / P/ 69 Investigation of the molecular mechanisms by which 97
Listeria monocytogenes grows in the mammalian gall bladder
Dowd, G., Joyce, S., Casey, P. G., Hill, C. and Gahan, C. G.

B / P/ 70 Role of cadmium efflux system in Listeria monocytogenes virulence 97
Camejo, A. and Cabanes, D.
B / P/ 71 Investigation of chitinase as a potential virulence factor 98
in Listeria monocytogenes
Chaudhuri, S.
B / P/ 72 Sub-lethal concentrations of common disinfectants do not 98
influence survival and growth of Listeria monocytogenes in whole blood
Holch, A., Gaedt Kastbjerg, V. and Gram, L.
B / P/ 73 Internalin LRR domain variability in Listeria monocytogenes 99
isolated from different hosts
Zaytseva, E. A., Ermolaeva, S. A. and Somov, G. P.
B / P/ 74 Reduced virulence of an adenylosuccinate lyase transposon mutant 99
of a serotype 4b strain of Listeria monocytogenes
Faith, N. G., Kim, J.-W., Kathariou, S., Sahaghian, R. and Luchansky, J. B.
B / P/ 75 Manifestations and outcome of listeriosis in adult patients 100
Fernández Guerrero, M L., Mancebo Plaza, B., Torres, R., Górgolas, M. and Jusdado, J. J.
B / P/ 76 Model for human Listeriosis: in vivo monitoring of orally infected mice 100
using bioluminescent Listeria monocytogenes
Bergmann, S., Lengeling, A., Pasche, B. and Schughart, K.
B / P/ 77 Galleria mellonella as model system to study Listeria species-specific 101
and Listeria monocytogenes serotype-specific pathogenesis
Mraheil, M. A., Krishnendu, M., Hain, T. and Chakraborty, T.
B / P/ 78 Listeria monocytogenes ActA is a key player in evading autophagic recognition 101
Pillich, H., Loose, M., Hain, T. and Chakraborty, T.
B / P/ 79 Non-haemolytic and hypovirulent Listeria monocytogenes became 102
haemolytic and virulent after passage through mice
Secic, I., Lindbäck, T. and Rørvik, L. M.
B / P/ 80 Mutants of Listeria monocytogenes (Lm) resistant to the polycationic 102
peptide protamine appear to be attenuated for virulence
Schlech, W.
B / P/ 81 The role of plasmacytoid dendritic cells in the course 103
of Listeria monocytogenes infection
Solodova, E., Lienenklaus, S., Jablonska, J. and Weiss, S.
B / P/ 182 Oxygen restriction increases the infection potential of 103
Listeria monocytogenes – a transcriptional analysis
Andersen, J. B., Bergstrøm, A., Knudsen, G., Bak Christensen, B., Ebersbach, T.,
Boye, M. and Rask Licht, T.
AREA //C // Epidemiologic and clinical aspects of Listeria monocytogenes
and listeriosis
PLENARY LECTURES
C / PL/ 07 Update on clinical aspects of listeriosis: What’s new? 25
Schlech, W. F.
C / PL/ 08 A Canadian outbreak of listeriosis due to deli-meat: 25
Driving change in the food safety system
Farber, J. M., Pagotto, F., Gilmour, M., Nadon, C., Savelli, C., and MacDonald, D.
C / PL/ 09 Listeria monocytogenes phagocytic strategy 26
Alvarez-Dominguez, C.
ORAL PRESENTATIONS
C / O/ 24 Human listeriosis due to Listeria ivanovii 45
Guillet, C., Join-Lambert, O., Le Monnier, A., Leclercq, A., Mamzer-Bruneel, M. F.,
Bielecka, M. K., Scortti, M., Disson, O., Vazquez-Boland, J., Lortholary, O. and Lecuit, M.
C / O/ 25 Foodborne Listeriosis in India: An update 45
Barbuddhe, S. B., Malik, S. V. S., Ashok Kumar, J., Kalorey, D. R., Kurkure, N. V.,
Rawool, D. B., Swain, B. K., Korikanthimath, V. S., Chakraborty, T.
C / O/ 26 Human listeriosis and co-morbidities in England, 1999 to 2008: 46
quantifying the risk
Mook, P., Grant, K., O’Brien, S. J. and Gillespie, I.
C / O/ 27 Risk factors for death in Listeria monocytogenes infection, 46
England and Wales, 1990 to 2008
Mook, P., Grant, K. and Gillespie, I.
C / O/ 28 A discrete event model to track Listeria monocytogenes 47
in the retail environment
Pouillot, R. and Gallagher, D.

C / O/ 29 Clinical and epidemiological aspects of listeriosis in Israel 47
Hershko-Klement, A., Eliav, H., Valinsky, L., Schechner, V., Braun, E., Paitan, Y.,
Block, C. S. and Nir-Paz, R.
C / O/ 30 Human isolates of Listeria monocytogenes during half a century in Sweden 48
Lopez-Valladares, G., Danielsson-Tham, M.-L., Vishal Singh, P. and Tham, W.
C / O/ 31 Estimated incubation periods for listeriosis vary according 48
to clinical form of disease
Goulet, V., King, L., Vaillant, V. and De Valk, H.
C / O/ 32 Listeria monocytogenes in food and animals in the European Union in 2008 49
da Silva Felício, M. T., Rizzi, V., Boelaert, F. and Makela, P.
C / O/ 33 Listeria monocytogenes infection in the over 60s in England 49
between 2005 and 2008: a retrospective case-control study utilising
market research panel data
Gillespie, I. A., Mook, P., Little, C. L. and Grant, K.
C / O/ 34 Better and faster typing, MLVA – shall we play together? 50
Larsson, J. T., Roussel, S. and Moller Nielsen, E.
C / O/ 35 comK prophage junction fragments in Listeria monocytogenes contain 50
SNPs that differentiate subclones of ECII and ECIII that are unique
to individual meat and poultry processing plants in the U.S.
Knabel, S. J.
C / O/ 36 Genetic diversity of Listeria monocytogenes measured by multiple-locus 51
variable-number tandem repeat analysis (MLVA)
Hyytia-Trees, E., Sabol, A., Graves, L. and Ribot, E.
POSTER PRESENTATIONS
C / P/ 82 Semi-automated repetitive sequenced-based PCR compared 104
to pulsed field gel electrophoresis for Listeria monocytogenes sub-typing
Roussel, S., Félix, B., Vignaud, M.-L., Tam Dao, T., Marault, M. and Brisabois, A.
C / P/ 83 Listeriosis: a frequent cause of fatal encephalitis in France 104
with high case fatality
Mailles, A., Vaillant, V., Lecuit, M. and Stahl, J.-P.
C / P/ 84 Listeria monocytogenes: identification and subtyping 105
Favretti, M.
C / P/ 85 Virulotyping of Listeria monocytogenes by high resolution melt analysis 105
Amar, C. F., Tamburro, M., Dear, P. and Grant, K.
C / P/ 86 Risk factors for nonperinatal listeriosis mortality in Los Angeles County, 106
California, 1992–2004
Guevara, R. E., Mascola, L. and Sorvillo, F.
C / P/ 87 Molecular typing of Listeria monocytogenes isolated from ovine sausage 106
Nives, M. R., Mele, P., Parisi, A., Latorre, L., Virgilio, S. and Tola, S.
C / P/ 88 A novel phage-PCR assay for the rapid detection of viable 107
Listeria monocytogenes in food within 24 h
Elemam, M. M.
C / P/ 89 Perinatal listeriosis in Los Angeles County, California, 1992–2004 107
Guevara, R. E. and Mascola, L.
C / P/ 90 Antimicrobial resistance of Listeria monocytogenes human strains 108
isolated since 1926 in France
Morvan, C., Moubareck, A., Leclercq, M., Herve-Bazin, S., Bremont, M.,
Lecuit, P. and Le Monnier Courvalin, A.
C / P/ 91 Today and tomorrow: The molecular epidemiology of listeriosis in the UK 108
Grant, K., Amar, C., Matos, J., Mook, P., Little, C. and Gillespie, I.
C / P/ 92 Use of Fluorescent Amplified Fragment Length Polymorphism 109
for improved typing of Listeria monocytogenes
Matos, J., Amar, C., Desai, M., Cross, L. and Grant, K.
C / P/ 93 Towards an application for field samples of a multipathogen platform 109
for molecular detection of raw milk pathogens
Omiccioli, E, Amagliani, G., Brandi, G., Tonucci, F., Foglini, M. and Magnani, M.
C / P/ 94 Detection and recovery of Listeria species from stainless steel 110
Boone, R., Iugovaz, I, Trottier, Y.-L. and Pagotto, F.
C / P/ 95 Natural carriage of Listeria in fresh-water fish in Russia 110
Egorova, I., Voronin, M., Selyaninov, Y. and Kolbasov, D.
C / P/ 96 Prevalence of Listeria in wild fauna in Russia 111
Egorova, I., Fertikov, V. and Kolbasov, D.
C / P/ 97 Quantitative assessment of the exposure to Listeria monocytogenes 111
from soft-ripened cheese consumption in North America:
a joint FDA/Health Canada project
Gendel, S., Pouillot, R., Murray, C., Farber, J., Ross, W., Couture, H. and Jean, A.

C / P/ 98 Human listeriosis in England, 2001 – 2007: association with 112
neighbourhood deprivation
Gillespie, I. A., Mook, P., Little, C. L., Grant, K. and McLauchlin, J.
C / P/ 99 Characterisation of antibodies for use in an antibody-based sensor 112
for on-site detection of L. monocytogenes
Gilmartin, N., Hearty, S. and O’ Kennedy, R.
C / P/ 100 Food Investigation of sporadic cases of neuroinvasive listeriosis 113
Goulet, V., Leclercq, A., Laurent, E., King, L., Dusch, V., Salem, S., Vaillant, V.,
Chenal-Francisque, V., de Valk, H. and Pihier, N.
C / P/ 101 Evaluation of the antimicrobial activity of Vaccinium myrtillus, 113
Prunus domestica and Myrtus communis against Listeria monocytogenes
Serio, A., Di Pasquale, F. and Paparella, A.
C / P/ 102 High throughput quantitative detection of Listeria monocytogenes 114
in food by RealTime PCR
Cammà, C., Ancora, M., Rizzi, V., Sperandii, A., Prencipe, V. and Migliorati, G.
C / P/ 103 Bibliographic study concerning procedures for preparing 114
environmental samples for analyses, regarding to L. monocytogenes
Barre, L., Carpentier, B. and Gnanou Besse, N.
C / P/ 104 Persistent L. monocytogenes isolates from Austrian and Irish dairies 115
show the same phenotypic and genetic background
Stessl, B.
C / P/ 105 Epidemiological data on listeriosis in Portugal: 2003 – 2008 115
Almeida, G, Magalhães, R., Hogg, T. and Teixeira, P.
C / P/ 106 Diversity among Listeria monocytogenes isolated from humans 116
Kalekar, S., Rodrigues, J., D’Costa, D., Malik, S. V. S., Kalorey, D. R.,
Chakraborty, T. and Barbuddhe, S. B.
C / P/ 107 Characterization of Listeria isolated from seafood 116
Rodrigues, J., Kalekar, S., Bhosle, S. N., Doijad, S. and Barbuddhe, S. B.
C / P/ 108 Characterization of Listeria species isolated from milk 117
D’Costa, D., Bhosle, S. N., Dhuri, R. B., Kalekar, S., Rodrigues, J., Doijad, S. P. and Barbuddhe, S. B.
C / P/ 109 Prevalence of Listeria monocytogenes in chicken production chain 117
in Thailand
Kanarat, S., Nijthavorn, N. and Sukhapesna, J.
C/P/110 Listeria monocytogenes in Ireland – Epidemiology and Molecular Typing 118
Cormican, M. G., DeLappe, N., McKeown, P. and Garvey, P.
C / P/111 Characterization of Listeria monocytogenes isolated 118
from human cases of listeriosis occurred in Portugal in 2008
Magalhães, R., Barbosa, J., Santos, I., Almeida, G. and Teixeira, P.
C / P/112 Post-processing environmental contamination of surface-ripened 119
soft cheese during affinage
D’Amico, D. and Donnelly, C.
C / P/113 Genetic diversity of Listeria monocytogenes isolated 119
from Portuguese cheeses
Almeida, G., Magalhães, R., Santos, I., Barbosa, J., Hogg, T. and Teixeira, P.
C / P/114 Prevalence of Listeria spp. in retail raw ground beef in Izmir, Turkey: 120
A comparison of standard cultural method and Fluorescent in situ
Hybridization (FISH) technique for detection
Handan Baysal, A.
C / P/115 Using ListexP100 for Listeria monocytogenes detection in foods 120
Flores Lopes, J., Ferreira Leite, I., Azeredo, J., Gibbs, P. and Teixeira, P.
C / P/116 Mapping of molecular profiles associated to Listeria monocytogenes 121
food isolates circulating in Italy
De Cesare, A., Parisi, A., Latorre, L. and Manfreda, G.
C / P/117 Zoonotic aspects of Listeria monocytogenes isolates 121
from zebu dairy animals
Parihar, V. S. , Barbuddhe, S. B., Kalorey, D. R., Kotwal, S.,Danielsson Tham, M.-L. and Tham, W.
C / P/118 Performance of ALOA and Palcam agars for detection of 122
Listeria monocytogenes in naturally contaminated raw meat products
Gravato Rowlands, R. E., Asturiano Ristori, C. A., Geraldes Martins, C.,
Jakabi, M. and Gombossy de Melo Franco, B. D.
C / P/119 Comparison of MOPS-BLEB and Fraser as secondary enrichment broths 122
for Listeria monocytogenes
Upham, J., Huszczynski, G., Mosher, M., Borza, A., Dorey, M., Bosley, J., Hara, K.,
Mutanda, C., Liu, J., Byrne, B. and Douey, D.
C / P/120 Comparison of UVM, Palcam and Oxoid Novel Enrichment (ONE) 123
broth as primary enrichment broths for Listeria monocytogenes
Upham, J., Mosher, M., Borza, A., Huszczynski, G., Dorey, M., Eloranta, K. and Douey, D.
C / P/121 Isolation and characterization of Listeria monocytogenes 123
from asazuke (Japanese light pickles)
Maklon, K., Kusumoto, A., Makino, S.-I. and Kawamoto, K.

C / P/122 Grouping of human Listeria monocytogenes isolates 124
Lopez-Valladares, G., Danielsson-Tham, M.-L. and Tham, W.
C / P/123 Characterization of Listeria monocytogenes strains isolated 124
from food and environmental samples
Nucera, D. M., Lomonaco, S., Manila Bianchi, D., Decastelli, L., Grassi, M. A. and Civera, T.
C / P/124 Development of a multiplex SNP-typing method to identify 125
the four epidemic clones of Listeria monocytogenes
Lomonaco, S., Civera, T., Dalmasso, A., Knabel, S. J. and Bottero, M. T.
C / P/125 Listeria monocytogenes incidents reported to the UK 125
Food Standards Agency from 2000 to 2008
Aish, J.
C / P/183 Genetic diversity of Listeria monocytogenes in broiler flocks 126
Courtillon, C. Toquin, M.-T., Le Nôtre, Y., Fravalo, P. and Mansour Chemaly, M.
C / P/184 Tracing Listeria monocytogenes contaminations throughout 126
the processing chain of a typical Italian pork meat product
using Pulsed Field Gel Electrophoresis (PFGE) characterization
Annunziata Prencipe, V., Acciari, V., Torresi, M., Migliorati, G.,
Marfoglia, C. and Valentina Rizzi, V.
C / P/185 Production and validation of capture-ELISA kit based on monoclonal 127
antibodies specific for Listeria monocytogenes in foodstuffs
Portanti, O., Di Febo, T., Luciani, M., Pompilii, C., Armillotta, G., Principe, V.,
Lelli, R. and Semprini, P.
C / P/186 Production of a reference material for microbiological tests 127
containing Listeria innocua
Pomilio, F., Ricci, L, Di Giannatale, E., Semprini, P., Candeloro, L. and Migliorati, G.
AREA //D // Strategies for prevention and control of Listeria monocytogenes
PLENARY LECTURES
D / PL/ 10 Risk assessment using the microbiological criteria: arguments for 27
zero tolerance (USDA) other viewpoint (Europe):
Risk-based microbiological criteria for Listeria monocytogenes
in RTE foods
Luber, P.
D / PL/ 11 USDA regulatory approach and considerations for the control 27
of Listeria monocytogenes
Engeljohn, D.
D / PL/ 12 Listeria monocytogenes, an emergent pathogen in Chile and Latin America 28
Hormazábal, J. C.
ORAL PRESENTATIONS
D / O/ 37 Time Temperature Indicators can be used as an effective 52
Risk Management Tool for Listeria monocytogenes in Ready-To-Eat foods
Koutsoumanis, K., Vaikousi, H. and Costas, B.
D / O/ 38 Safety of Salad Leaves and Herbs 52
Garland, C. D. and Clark, A.
D / O/ 39 Environmental factors affect adhesion of Listeria monocytogenes 53
to inert surfaces through flagellum expression
Tresse, O.
D / O/ 40 Shelf-life laboratory durability and challenge studies for Listeria 53
monocytogenes in ready-to-eat foods: a presentation of the
European technical guidance document intended for laboratories
Beaufort, A., Bergis, H., Cornue, M. and Lardeux, A.-L.
D / O/ 41 Successful strategies against Listeria monocytogenes in Switzerland 54
Imhof, R.
D / O/ 42 Absence of Listeria monocytogenes growth during raw milk cheesemaking: 54
a modelling approach
Jordan, K., Schvartzman, S. Butler, F. and Tenenhaus-Aziza, F.
D / O/ 43 A predictive model to set high pressure processing criteria 55
for Listeria monocytogenes inactivation on dry-cured ham
Bover-Cid, S., Belletti, N., Garriga, M. and Aymerich, T.
D / O/ 44 Application of a validated predictive model to prevent growth 55
of Listeria monocytogenes in ready-to-eat foods – importance
for product development and risk management
Mejlholm, O. and Dalgaard, P.
D / O/ 45 Application of predictive microbiology to control the growth 56
of Listeria monocytogenes – dairy products as an example
Lobacz, A.

D / O/ 46 Characterization of Food Alert for Listeria monocytogenes in France in 2008 56
Leclercq, A., Dusch, V., Salah, S., Laurent, E., Chenal-Francisque, V.,
Thierry-Bled, F., Lecuit, M., Goulet, V. and Pihier, N.
POSTER PRESENTATIONS
D / P/ 126 Colonization of a newly constructed commercial chicken further 128
processing plant with Listeria monocytogenes
Berrang, M. E., Meinersmann, R., Frank, J. and Ladely, S.
D / P/ 127 Episcopic differential interference contrast/epifluorescence microscopy 128
to characterise in situ Listeria monocytogenes biofilms
on stainless steel surfaces
Gião, M. S. and Keevil, C. W.
D / P/ 128 Temperature dependent defect in biofilm formation 129
by Listeria monocytogenes
Abdalla, S., Glenn, S., Shama, G. and Andrew, P.
D / P/ 129 Mode of action of Lactococcus lactis sa31 antimicrobial peptide 129
on Listeria monocytogenes ½ c
Barile, M., Mormile, A., Ceres, C., Pepe, O., Cortesi, M. L. and Murru, N.
D / P/ 130 Tracing the source, epidemiology and persistence of Listeria monocytogenes 130
in Irish Farmhouse cheese processing facilities
Jordan, K., Fox, E., O’Brien, M. and Hunt, K.
D / P/ 131 Phenotypic and genotypic characteristics of Listeria monocytogenes 130
strains isolated from a convenience food-processing plant
Blatter, S., Stephan, R., Tasara, T. and Zweifel, C.
D / P/ 132 Influence of flow direction on the adhesion of Listeria monocytogenes 131
to brushed stainless steel surfaces
Skovager, A., Whitehead, K., Ingmer, H., Verran, J. and Arneborg, N.
D / P/ 133 Occurrence of Listeria monocytogenes in raw milk and dairy products 131
in Kazerun, Iran
Mehdi Mahmoodi, S. M. and Javanmardi, F.
D / P/ 134 Antilisterial mode of action of bacteriocin ST182Gu produced 132
by Enterococcus casseliflavus isolated from guava
Todorov, S., Destro, M. T., Chiarini, E. B., Vaz-Velho, M. and Franco, B. D. G. M.
D / P/ 135 Control of Listeria monocytogenes in fresh goat cheese by bacteriocinogenic 132
strain Lactococcus lactis subsp. lactis DF4Mi or commercial nisin
Nader Furtado, D., Todorov, S., Landgraf, M., Destro, M. T. and Franco, B. D. G. M.
D / P/ 136 High pressure processing ensures elimination of Listeria monocytogenes 133
in sliced dry cured ham
Stollewerk, K., Jofré, A., Comaposada, J., Aymerich, T., Ferrini, G., Arnau, J. and Garriga, M.
D / P/ 137 Inhibition of Listeria monocytogenes by Lactococcus sp. EU2241 133
in tropical shrimp
Abdoulaye Fall, P.
D / P/ 138 Listeria monocytogenes and Listeria innocua in slaughter line of 134
a swine meatpacking plant in Rio Grande do Sul State, Brazil
Schittler, L. and Padilha Silva, W.
D / P/ 139 Listeria monocytogenes in raw meat products marketed in the city 134
of Sao Paulo, Brazil: Incidence and counts data for risk assessment
Ristori, C. A., Rowlands, R. E. G., Martins, C. G., Fávero, L. M. and Franco, B. D. G. M.
D / P/ 140 Inhibiton of Listeria monocytogenes by Carnobacterium maltaromaticum 135
in combination with extract of Lippia sidoides Cham.
in cold-smoked surubim fish broth
Barbosa dos Reis, F., de Souza, V. M., Sousa Thomaz, M. R., Pinto Fernandes, L.,
Pereira de Oliveira, W. and Pereira De Martinis, E. C.
D / P/ 141 Inhibition of Listeria monocytogenes in cooked ham 135
by virulent bacteriophages and protective cultures
Holck, A., Schirmer, B. C. and Berg, J.
D / P/ 142 Surface colonization by Listeria monocytogenes: role of the flagella 136
in the biofilm formation process
Desvaux, M., Briandet, R., Renier, S., Deschamps, J., NCaccia, N., Chafsey, I. and Hébraud, M.
D / P/ 143 The role of sanitizers in controlling Listeria monocytogenes 136
on stainless steel surfaces: Lessons learned
from the 2008 Listeriosis outbreak
Hébert, K., Farber, J. and Pagotto, F.
D / P/ 144 Listeriophage ecology and diversity on dairy farms 137
Vongkamjan, K., Moreno Switt, A., den Bakker, H. C., Fortes, E. D., and Wiedmann, M.
D / P/ 145 Evaluation of curative and preventive decontamination treatments 137
on Listeria monocytogenes biofilms with a new screening system
Quinon, E., Chamot, S., Groelly, J., Chavant, P., Bernardi, T., Desvaux, M. and Hebraud, M.

D / P/ 146 Pulsed Field Gel Electrophoresis, conventional and molecular 138
serotyping on Listeria monocytogenes: An European Proficiency
Testing Inter-laboratory Trial
Félix, B., Roussel, S., Tam Dao, T., Asséré, A., Lombard, B. and Brisabois, A.
D / P/ 147 Detection of Listeria spp. in raw and pasteurized liquid egg-products 138
and in the egg-breaking plants environment
Rivoal, K., Fablet, A., Chemaly, M., Salvat, G. and Protais, J.
D / P/ 148 Biofilm formation by Listeria monocytogenes isolates under conditions 139
that mimic food and digestive tract
Kretli Winkelströter, L., Oliveira, M. A. and De Martinis, E. C.
D / P/ 149 Biofilm formation of Listeria monocytogenes EGDe depends 139
on temperature and nutrient availability
Auchter, M., Endres, J., Waidmann, M. S. and Riedel, C. U.
D / P/ 150 Antimicrobial activity of lactococcal and enterococcal strains 140
isolated from artisanal products from North West of Italy
toward Listeria monocytogenes
Dal Bello, B., Rantsiou, K., Ambrosoli, R., Zeppa, G. and Cocolin, L.
D / P/ 151 Plant-based strategies for Listeria monocytogenes control in foods 140
Paparella, A., Serio, A., Chaves-Lopez, C. and Di Pasquale, F.
D / P/ 152 Scientific studies for survival of Listeria monocytogenes 141
in dairy products and its application in practice
Cabanova, L., Škuntova, O. and Kantikova, M.
D / P/ 153 The effect of chilling temperatures on the virulence 141
of Listeria monocytogenes isolates with different origins
Neves, E. M., Silva, A. C., Louro, P., Ferreira-Dias, S. and Brito, L.
D / P/ 154 How to improve a sampling plan in order to better assess 142
L. monocytogenes contamination on diced bacon at the plant
Bergis, H., Commeau, N., Zuliani, V., Cornu, M., Beaufort, A. and Garry, P.
D / P/ 155 Contamination of Listeria monocytogenes in a cold-smoked pork 142
processing plant using brining injections
Berzins, A., Silins, I. and Korkeala, H.
D / P/ 156 Effects of GRAS products on growth of Listeria monocytogenes 143
during cold storage of salmon fillets
McCarthy, S. and Johnson, D.
D / P/ 157 Heavy-metal and detergent resistance of Listeria species isolates 143
from milk processing environments
Doijad, S., Garg, S. and Barbuddhe, S. B.
D / P/ 158 Detection of Listeria monocytogenes in lettuce sold at markets 144
and supermarkets in Porto, Portugal
Noronha, L., Silva, J. and Teixeira, P.
D / P/ 159 Preliminary analysis of structure and chemical composition of 144
extracellular polymeric substance produced by Listeria monocytogenes
Nwaiwu, O., Lad, M., Davis, A., Foster, T. and Rees, C.
D / P/ 160 Ecology and persistence of Listeria monocytogenes strains in fermented 145
meat sausage processors from the Northern region of Portugal
Ferreira, V., Barbosa, J., Vongkamjan, K., Moreno Switt, A., Hogg, T., Gibbs, P.,
Wiedmann, M. and Teixeira, P.
D / P/ 161 Biofilm formation and survival of L. monocytogenes and slaughter house 145
bacteria on surfaces at relevant environmental conditions
Langsrud, S., Møretrø, T. and Heir, E.
D / P/ 162 Control of L. monocytogenes by lysozyme combined with olive leaf extract 146
in edible pullulan film coated on chicken breast fillets
Handan Baysal, A.
D / P/ 163 Evaluation of antilisterial activity by lactic acid bacteria 146
Borges, S., Barbosa, J., Albano, H., Silva, J. and Teixeira, P.
D / P/ 164 Molecular methods to assess Listeria monocytogenes route 147
of contamination in a dairy processing plant
Cocolin, L., Alessandria, V., Dolci, P. and Rantsiou, K.
D / P/ 165 Persistence of L. monocytogenes in artisanal cheese producing plants 147
Almeida, G., Santos, I., Magalhães, R., Barbosa, J., Hogg, T. and Teixeira, P.
D / P/ 166 Occurrence of Listeria monocytogenes in food products collected 148
in Portugal from retail establishments and food plants
Mena, C., Carneiro, L., Santos, I., Magalhães, R., Almeida, G. and Teixeira, P.
D / P/ 167 Listeria monocytogenes biofilms grown at 12 °C showed reduced 148
susceptibility to sanitizers
Afonso Lourenço, A., Machado, H. and Brito, L.
D / P/ 168 Modelling growth of Listeria monocytogenes in cheese as function 149
of environmental variables
Sand Rosshaug, P. and Hallberg Larsen, M.
D / P/ 169 Characterization of anti-Listerial bacteriocin produced by 149
Lactobacillus plantarum ST8SH, a strain isolated from Bulgarian salami
Todorov, S. D. and Lemos Vaz-Velho, M.

D / P/ 170 EFSA’s proposal for an EU-wide retail survey on Listeria monocytogenes 150
in selected categories of ready-to-eat food products
Frank Helena Boelaert, F. H., Felício, T. and Makela, P.
D / P/ 171 Ripening conditions: an asset to control L. monocytognes in cheeses 150
Callon, C., Picque, D., Corrieu, G. and Montel, M.-C.
D / P/ 172 Deterministic and stochastic behavior of Listeria monocytogenes 151
suspended cells or detached from stainless steel surfaces
during cheese manufacturing
Belessi, C.-E. A., Gounadaki, A. S., Arapakh, S., Schvartzman, S., Jordan, K. and Skandamis, P. N.
D / P/ 173 Prevalence of Listeria monocytogenes in game meat 151
Atanassova, V.
D / P/ 174 Relationship between pathogenic profile and in vitro biofilm formation 152
capacity of Listeria monocytogenes strains isolated from meat,
fish and processing plants
Meloni, D., Mazza, R., Marceddu, M., Piras, F., Mureddu, A. and Mazzette, R.
D / P/ 175 Prevalence and molecular characterization of Listeria monocytogenes 152
in traditional fermented pork sausages produced in Italy
Mazzette, R., Meloni, D., Busia, G., Melillo, R., Mureddu, A. and Piras, F.
D / P/ 176 Minimum Biofilm Eradication Concentration (MBEC) of different 153
antimicrobials on Listeria monocytogenes and Salmonella enterica biofilms
Rodrigues, D., Teixeira, P., Oliveira, R., Ceri, H. and Azeredo, J.
D / P/ 177 Examination of the ability of adherence, biofilm formation and sensitivity 153
to some disinfectants of different Listeria monocytogenes strains
Milanov, D., Vidić, B., Petrović, J., Bugarski, D. and Ašanin, R.
D / P/ 187 Evolution of Listeria monocytogenes contamination in poultry production: 154
from the farms to the processing levels
Mansour Chemaly, M., Toquin, M.-T., Courtillon, C., Le Nôtre, Y., Rivoal, K. and Fravalo, P.
D / P/ 188 A regular survey of Listeria in ready-to-eat foods (2004 – 2009) 154
Furtado, R., Loreto Campos, M., Correia, C., Ferreira, I., Maia, C., Rosa, N., Santos, S.,
Santos, M. I. and Saraiva, M.
D / P/ 189 Incidence of Listeria monocytogenes in Queijo Fresco 155
Rosa, N., Campos, L., Correia, C., Ferreira, I., Furtado, R., Maia, C., Santos, S.,
Cunha, C. I. and Santos, M. I.
D / P/ 190 Risk factors for Listeria monocytogenes contamination 155
in French broiler flocks
Aury, K., Le Bouquin, S., Toquin, M.-T., Petetin, I., Le Nôtre, Y., Allain, V.,
Fravalo, P. and Mansour Chemaly, M.
D / P/ 191 Portuguese sushi: is it contaminated with L. monocytogenes? 156
Mendes, D., Furtado, R., Maia, C., Correia, C., Campos Cunha, I., Pedroso, L. and Santos, M. I.
D / P/ 192 Effect of the inoculum size on growth of L. monocytogenes 156
in dices of poultry breast
Lardeux, A.-L., Gnanou-Besse, N., Doux, C. and de Courseulles, E.
D / P/ 193 Investigation into the mechanisms of detergent induced changes 157
in disinfectant susceptibility of attached Listeria monocytogenes
Walton, J., Hayes, R., Protheroe, R., Hill, D. and Gibson, H.
D / P/ 194 Prevalence of Listeria monocytogenes throughout the production process 157
of Parma ham: tracing contaminations from slaughterhouses
to the final product
Prencipe, V. A., Rizzi, V., Iannetti, L., Serraino, A., Calderone, D., Rossi, A., Morelli, D.,
Marino, L. and Migliorati, G.
D / P/ 195 Prevalence of Listeria monocytogenes in raw milk sold at vending machines 158
in Abruzzo region
Prencipe, V. A., Scattolini, S., Sperandii, A. F. and Migliorati, G.
D / P/ 196 Characterization of Listeria monocytogenes strains isolated from soft 158
and semi soft cheeses sampled at retail level
Acciari, V., Torresi, M., Migliorati, G., Di Giannatale, E., Semprini, P. and Prencipe, V.
D / P/ 197 Preliminary report on the organisation of a food microbiology 159
proficiency testing program as a tool to guarantee the equivalence
of the US and IT official control systems
Di Giannatale, E., Marfoglia, C., Prencipe, V., Salini, R., Migliorati, G. and Ricci, L.
D / P/ 198 High nisin susceptibility of Listeria spp. wild-type strains isolated 159
from dairies with traditional cheese preservation in Portugal
Pintado, C. M. B. S and Ferreira M. A. S. S.
D / P/ 199 Utilization of Lactococcus lactis M104, a wild nisin-producing 163
raw milk isolate, as an antilisterial adjunct in traditional
Greek Graviera cheese processing
Samelis, I., Pappa, E., Bogovic-Matijasic, B. and Rogelj, I.
D / P/ 200 The European Project BASELINE “Selection and improving 163
of fit-for-purpose sampling procedures for specific foods and risks”
Manfreda, G. and De Cesare, A.

AREA //E // Communication, risk perception and consumer practices –
Social sciences in Listeria control
PLENARY LECTURES
E / PL/ 13 How can the social sciences help us understand the prevalence of 29
listeriosis in the UK?
Wadge, A.
E / PL/ 14 Consumer perceptions, behaviour and microbial food safety; 29
implications for Listeria control
Frewer, L. J.
ORAL PRESENTATIONS
E / O/ 47 Efforts to update and perform health education on listeriosis in 57
Los Angeles County, California, by the County of Los Angeles Department
of Public Health
Guevara, R. E.
E / O/ 48 Awareness of listeriosis among Portuguese pregnant women 57
Mateus, T., Maia, R. L. and Teixeira, P.
E / O/ 49 The development and progress of a risk-based strategy 58
for the control of Listeria monocytogenes in New Zealand
Castle, M. and Crerar, S.
POSTER PRESENTATIONS
E / P/ 178 What is an appropriate level of protection for Listeria monocytogenes 160
in foodstuffs consumed by vulnerable groups?
Little, C., Gillespie, I., Grant, K., Gormley, F., Mook, P. and McLauchlin, J.
E / P/ 179 ILCD: An Interactive Listeria culture diversity knowledgebase 160
Ashok Kumar, J., Barbuddhe, S. B., Kalekar, S., Rodrigues, J., Chopade, N. A.,
Hain, T. and Chakraborty, T.
E / P/ 180 Listeria spp. and the domestic environment: consumer knowledge, 161
attitudes, risk perceptions and food-handling behaviours
Redmond, E. C.
E / P/ 181 Do you know the temperature in your refrigerator? 161
Røssvoll, E., Jacobsen, E., Ueland, Ø., Einar Granum, P. and Langsrud, S.
Authors Index 165

PLENARY LECTURES

KEY NOTE SPEACH
Listeria monocytogenes: a multifaceted model
Cossart, P.*
Institut Pasteur, Unité des Interactions Bactéries cellules, Inserm U604, INRA USC2020
Listeria monocytogenes is an intracellular pathogen responsible for severe human
food-borne infections characterized by gastroenteritis, materno-fetal infections
and brain infections with a mortality rate of 30 %. The disease is mainly due the capacity
of Listeria to cross three host barriers: the intestinal barrier, the maternofetal
barrier and the blood brain barrier. It is also due to the capacity of the organism
to survive and replicate in macrophages and to enter and replicate in non
phagocytic cells. We use a combination of approaches to understand the mechanisms
which allow establishment and maintenance of a Listeria infection. We then
investigate if our findings have a general significance. In nearly three decades of
molecular and cellular investigations, the study of Listeria momocytogenes and of
listeriosis has led to new concepts in infection biology and in several other areas of
biology including cell biology, microbiology, molecular medicine and genomics.
* Plenary Supported by FCT
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AREA // A // Biology of Listeria monocytogenes
PLENARY LECTURES // PL/ 1–2
The pangenome of Listera spp.
Chakraborty, T.*
Institute of Medical Microbiology, Justus-Liebig University of Giessen, Germany
Listeria monocytogenes is a food-borne pathogen with a high mortality rate that
has served as an invaluable model for intracellular parasitism. We have sequenced
representative genomes comprising all species for the genus Listeria as well as
strains representing clonal lineages of the pathogenic species of L. monocytogenes.
Comparative genome analysis now provides clear evidence indicating that the various
non-pathogenic species of Listeria have been derived by gene loss and/or mutational
decay of virulence- and niche-adaptive factors from a progenitor strain
that harboured many of the currently known virulence factors. Thus, non-pathogenic
Listeria are compromised with regard to their ability to live in the cytoplasm
of infected host cells but have acquired genes enabling their growth in soil and decaying
vegetation. Comparative transcriptome analysis of intracellular growth has
also uncovered additional levels of adaptive evolution in growth among the different
lineages of L. monocytogenes. Analysis of the pan-genome of L. monocytogenes
strains revealed an extensive, as yet unexplained gene-repertoire in these
genomes, and provides evidence for the evolution of these strains by gathering
genes from organisms in different environmental and host niches.
* Plenary Supported by FCT
Ecology of L. monocytogenes and Listeria spp. in natural
and food associated environments
Wiedmann, M.*
Department of Food Science, Cornell University, Ithaca, NY, USA
Listeria spp., including the pathogen L. monocytogenes, can be isolated from a variety
of environments, often at considerable frequency. A number of our studies
have specifically shown that Listeria spp. can often be isolated from > 20 % of samples
collected from natural, urban, and farm environments and can also be commonly
isolates from food associated environments (e.g., processing plants, retail
environments). Molecular characterization and subtyping tools not only typically
reveal considerable diversity among Listeria spp. isolates from different environments,
but also provide evidence for (i) association between certain subtypes or
species and specific environments (suggesting existence of specific Listeria ecotypes)
as well as (ii) long-term persistence of specific Listeria strains in different
environments. For example, in one of our studies, L. seeligeri and L. welshimeri
were significantly associated with natural environments (p

AREA // B // Listeria monocytogenes as a human and animal pathogen
PLENARY LECTURES // PL/ 3–6
How Listeria monocytogenes breaches host barriers
Lecuit, M.*
Institut Pasteur, Inserm, Paris, France
Listeria monocytogenes (Lm) is a human foodborne pathogen that causes listeriosis,
a systemic infection leading to meningitis, encephalitis and feto-placental infection.
To reach its target organs, Lm crosses the intestinal, blood-brain and placental
barriers. We will present the results of our investigations regarding the
molecular mechanisms underlying Lm crossing of these three barriers.
* Plenary Supported by FCT
Secretion of a novel L. monocytogenes cyclic dinucleotide into the
cytosol of infected host cells activates an innate immune pathway
Portnoy, D. A.*
Department of Molecular and Cell Biology and The School of Public Health, University of California,
Berkeley, USA
Listeria monocytogenes has been used for decades as a model to study basic aspects
of intracellular parasitism and cell-mediated immunity. Because L. monocytogenes
induces a robust CD8+ T-cell response, attenuated strains of L. monocytogenes
are being developed as live, attenuated vaccine vectors for infectious
disease and malignancies. What makes L. monocytogenes such a strong inducer of
cellular immunity? As everyone in this audience appreciates, virulent strains of
L. monocytogenes secrete a pore-forming cytolysin (LLO) that allows bacteria access
the host cell cytosol. We previously discovered that vacuolar and cytosolic
bacteria induced distinct host transcriptional responses, the latter leading to the
expression of beta interferon and a host of co-regulated genes. To understand the
microbial components that activate the cytosolic pathway, we used a forward genetic
screen to identify bacterial mutants that induced an enhanced or diminished
host transcriptional response to cytosolic bacteria. Most of the mutants identified
mapped to genes encoding or regulating multidrug efflux pumps. We now have
a series of strains that induce beta interferon over a 60-fold range. These data
were consistent with a model in which a small molecula is either actively or inadvertently
being pumped from cells, and that the host can recognize and respond.
Using conventional biochemistry and mass spectrometry, we have identified the
L. monocytogenes molecule as a cyclic dinucleotide. The precise structure of this
novel bacterial molecule, identification of the cyclase and phosphodiesterase will
be presented at the meeting.
* Plenary Supported by FLAD
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AREA // B // Listeria monocytogenes as a human and animal pathogen
PLENARY LECTURES // PL/ 3–6
Diagnosis and clinical management of listeriosis
in ruminants and camelids
Poulsen, K. P.*
School of Veterinary Medicine, University of Wisconsin, Madison, USA
Listeria monocytogenes is a successful intracellular pathogen of domestic and food
producing animals. Ruminant (cattle, sheep, goats) and camelid (llama and alpaca)
species are constantly exposed to this gram-positive pathogen. It is ubiquitous in
the environment and tends to overgrow in poorly prepared silage (pH > 5.0). Listeriosis
manifests in these species as three distinct clinical syndromes including
abortion, neonatal sepsis, and meningioencephalitis (circling disease). Infection in
food producing animals, and subsequent shedding of L. monocytogenes in milk and
meat represents a significant risk to food safety. In this review, clinical signs, diagnostics,
and treatment of listeriosis of ruminant and camelid species will be presented
including video clips of clinically affected animals.
* Plenary Supported by FLAD
Listeriolysin S – a second haemolysin with a role in the virulence
of Listeria monocytogenes
Hill, C.*
Alimentary Pharmabiotic Centre and Microbiology Department, University College Cork, Ireland
Listeria monocytogenes require listeriolysin O (encoded by llo or hlyA) to escape
the vacuole and initiate intracellular growth and intercellular spread. Llo - mutants
are essentially avirulent and so listeriolysin O is regarded as the primary virulence
factor in L. monocytogenes. Given that all strains and serotypes possess this
virulence factor, it is unlikely to explain the increased incidence of particular
serotypes (such as 4b) in listeriosis epidemics. Many laboratories have sought to
solve the Listeria conundrum of why certain strains may be more likely to cause
disease in humans. We have identified a second haemolysin, which we have named
listeriolysin S, which is associated with epidemic strains. Listeriolysin S is very
different from listeriolysin O in that it is a highly modified peptide, which resembles
(in probable structure if not in primary sequence) the streptococcal virulence
factor, streptolysin S. In fact, a family of these modified virulence peptides can be
inferred from genomic data of other pathogens, including Staphylococcus aureus
and Clostridium botulinum. listeriolysin S is not expressed under laboratory conditions,
but can be induced with a number of reagents in vitro. In addition, a mutant
in which listeriolysin S is constitutively expressed is hyper-hemolytic in comparison
to wildtype strains. We have also demonstrated a small, but significant,
role for listeriolysin O in murine models of infection and in polymorphonuclear
leucocyte survival assays. Listeriolysin S provides a possible explanation for the
enhanced virulence of certain strains in human listeriosis, but much remains to be
done to confirm or refute this hypothesis.
* Plenary Supported by FEMS

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
PLENARY LECTURES // PL/ 7–9
Update on clinical aspects of listeriosis: What’s new?
Schlech, W. F.*
Dalhousie University, Canada
Listeriosis remains a challenging problem for clinicians. Diagnosis may be delayed
and treatments prolonged although decreases in mortality have been noted in
some surveillance programs. New syndromes, such as necrotizing fasciitis, have
been described as well as further outbreaks of febrile gastroenteritis. Sepsis and
rhomboencephalitis remain the most common presenting syndromes for invasive
listeriosis. Genetic markers for infection have also been uncovered. Infection in
the immunocompetent host still occurs but risk factors for the disease primarily
points to abnormalities in cell-mediated and innate immunity as major predispositions
to listeriosis. Use of TNF-alpha inhibitors and corticosteroids are of particular
concern. Some newer antibiotics active against L. monocytogenes have been
studied but ampicillin and gentamicin remains the treatment of choice. There is
further evidence that trimethoprim-sulfamethoxazole has a strong protective effect
against listeriosis in the compromised host receiving this drug for protection
against other pathogens.
* Plenary Supported by FCT
A Canadian outbreak of listeriosis due to deli-meat:
Driving change in the food safety system
Farber, J. M. 1*, Pagotto, F. 1, Gilmour, M. 2, Nadon, C. 2, Savelli, C. 3, and MacDonald, D. 3
1. Food Directorate, Health Canada
2. National Microbiology Laboratory, Public Health Agency Canada
3. Centre for Food-borne, Environmental and Zoonotic Infectious Diseases,
Public Health Agency Canada
In 2008, Canada experienced the first multi-provincial and largest outbreak of invasive
listeriosis. During this outbreak, 57 cases of illness, in 7 provinces, resulted
in 23 deaths from listeriosis, which was traced back to contaminated deli-meat
from Company A. The age range was 29 to 98 years, with the median age being 78.
All of the cases with a known medical history prior to exposure had underlying
medical conditions. Fifty (88 %) cases reported deli-meat consumption. Additionally,
84 % of cases had institutional exposure. Investigators confirmed Company A
deli meat was served to 27 cases. The major probable cause of the Listeria contamination
in Company A’s plant was related to meat slicing equipment. High-throughput
genome sequencing of two serotype 1/2a Listeria monocytogenes isolates was
completed during the outbreak. By screening for genetic traits specific to the outbreak
genomes, examination of clinical, environmental and food isolates associated
with the outbreak revealed that three distinct, but highly-related strains may have
been involved in this nationwide outbreak. As a result of the outbreak, the Public
Health Agency of Canada (PHAC), the Canadian Food Inspection Agency (CFIA)
and Health Canada (HC) conducted independent lessons-learned exercises. In addition,
the CFIA developed mandatory new Directives for federally-registered meat
and poultry plants, and Health Canada started updating their policy on Listeria
monocytogenes in RTE foods. Furthermore, a federal review by an independent investigator
(Ms. Sheila Weatherill) into the outbreak resulted in 57 recommendations
directed at food processors, regulators, public health professionals and individual
consumers. Work is currently underway at PHAC, HC and CFIA to directly
address the recommendations coming out of the Weatherill report.
* Plenary Supported by FCT
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AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
PLENARY LECTURES // PL/ 7–9
Listeria monocytogenes phagocytic strategy
Alvarez-Dominguez, C.*
Hospital Santa Cruz de Liencres-IFIMAV and IES Zapaton, Spain
Listeria monocytogenes enters macrophages and resides for a short period within
the phagosomal compartment before escaping and replicating in the cytosol. Listeria
has evolved a fine phagosomal strategy to survive the microbicidal machinery
of macrophages and avoid eliciting a strong innate immune response. First,
Listeria-GAPDH enzymatic modification and inactivation of Rab5a caused a delay
on phagosome maturation. Second, Listeria interferes with the trafficking of two
lysosomal proteins, cathepsin-D and LIMP2. Cathepsin-D enzymatic action blocks
the pore-forming function of Listeria-LLO within the phagosomes. Therefore, inhibiting
the transport of this lysosomal protease to the phagosomes increases Listeria
viability. Finally, the Listeria interference with LIMP2 trafficking disrupts
the connection between late endosomal events and the onset of innate immunity.
In brief, Listeria phagosomal strategy has the purpose to avoid the phagosomal
transformation into fully competent bactericidal and antigen-processing compartments.
Deciphering Listeria phagosomal strategy to subvert the host immune
response might help to design better therapies against listeriosis.
* Plenary Supported by FCT

AREA // D // Strategies for prevention and control of Listeria monocytogenes
PLENARY LECTURES // PL/ 10–12
Risk assessment using the microbiological criteria:
Arguments for zero tolerance (USDA) other viewpoint (Europe):
Risk-based microbiological criteria for Listeria monocytogenes
in RTE foods
Luber, P.*
Federal Office of Consumer Protection and Food Safety, Berlin, Germany
The control of Listeria monocytogenes in foods has been a focus activity ever since
the link between listeriosis in vulnerable populations and exposure via foods became
clear. Listeria spp. originate from soil and can contaminate many different
foods during food production or via processing environments. Once Listeria bacteria
get in a food plant, they might survive in the processing environment over
long time periods and could pose a contamination hazard to produced foods. Moreover,
the ability of L. monocytogenes to multiply under refrigeration temperatures
and without oxygen, for example in vacuum packs or in foods which are packed in
modified atmospheres, makes it challenging to control the bacteria further up in
the food chain at the retail level and once foods have reached consumers’ homes.
Risk assessments done in the past years have shown that amongst the many foods
which can become contaminated with L. monocytogenes, ready-to-eat (RTE) foods
which are consumed without further heat treatment are associated with the greatest
listeriosis risk. Regulators in the European Community and the Codex Alimentarius
Commission have developed risk-based microbiological criteria for L. monocytogenes
in foods. In both cases, the microbiological criteria specifically apply to
RTE foods and take into consideration if growth of Listeria in the food may occur
or not. However, in both approaches the main focus is on controlling L. monocytogenes
during processing of RTE foods and in the production environment. The European
Regulation on microbiological criteria (Regulation (EC) no 2073/2005) is
embedded in the so called ‘hygiene package’ of legislations, and in the Codex Alimentarius
microbiological criteria are presented in an Annex of the ‘Guidelines
on the Application of General Principles of Food Hygiene to the Control of Listeria
monocytogenes in Ready-to-Eat foods’ (CAC/GL 61-2007), only. In the European
Community, food business operators have an obligation to demonstrate compliance
with microbiological criteria and thereby enable verification of their GMP
and HACCP systems. The approach of using risk-based microbiological criteria
for L. monocytogenes as management option for controlling Listeria in foods and
thus to reduce the likelihood of listeriosis in vulnerable populations will be critically
discussed.
* Plenary Supported by FCT
USDA regulatory approach and considerations for the control
of Listeria monocytogenes
Engeljohn, D.*
U.S. Department of Agriculture, Food Safety and Inspection Service, Deputy Assistant Administrator,
Office of Policy and Program Development, USA
An overview of the design of the risk management design and objectives of the
control programs for ready-to-eat meat and poultry will be presented. Use of risk
assessments to inform the verification and enforcement strategy will be highlighted.
Lessons learned regarding the implementation of a “zero tolerance” approach
will be discussed, along with future plans for ensuring that progress towards
good control are not negated in further processing operations.
* Plenary Supported by FLAD
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AREA // D // Strategies for prevention and control of Listeria monocytogenes
PLENARY LECTURES // PL/ 10–12
Listeria monocytogenes, an emergent pathogen
in Chile and Latin America
Hormazábal, J. C.*
Public Health Institute of Chile
Food borne diseases are a major health burden in Latin America. In Chile Listeria
monocytogenes is a pathogen under laboratory surveillance since 2004. All the isolates
from human cases must be confirmed at the National Reference Laboratory
at the Public Health Institute of Chile. Until 2008 L. monocytogenes was an infrequent
pathogen, approximately 45 clinical cases per year were reported in all the
country. At the ending of 2008 an unusual increase of listeriosis was detected in
the Metropolitan region of Chile. In this scenario, the Reference Laboratory included
for first time molecular tools for the detection of related cases. PFGE including
PulseNet International protocols, was a powerful upgrade for the surveillance
system. In parallel The Ministry of health, in absence of a specific regulatory
framework for L. monocytogenes in food industry, made important changes in sanitary
food regulations, including specific microbiological criteria for Listeria in
food and environment. The integration of surveillance data, clinical, environmental
and food industry isolates, allowed the creation of a single database, including
molecular typing information. This dynamic system allowed the detection of the
first Chilean outbreak of L. monocytogenes, and made possible the link of clinical
cases to potential sources, including the environment, raw or processed food products.
After few weeks of an intensive investigation the outbreak source was confirmed
(goat and brie cheese). The inclusion of molecular tools in surveillance
provides valuable information for the early detection of listeriosis outbreaks.
PulseNet Latin America Network gives an important support for the construction
of national and regional databases for the detection of local outbreaks and virulent
clones with potential international spread. In Latin America Listeria keeps as an
infrequent and unknown pathogen, major efforts are necessary specially in health
staff and general population education including a sensitive surveillance system
and a strong regulatory framework in food industry.
* Plenary Supported by FCT

AREA // E // Communication, risk perception and consumer practices – Social sciences in Listeria control
PLENARY LECTURES // PL/ 13–14
How can the social sciences help us understand the prevalence
of listeriosis in the UK?
Wadge, A.*
Chief Scientist, Food Standards Agency, UK
There has been a marked change in the epidemiology of listeriosis in humans in the
UK over the past decade. A doubling of reported cases has been seen in England and
Wales since 2001 and this has occurred largely in people aged 60 years and over and
presenting with bacteraemia rather than central nervous system infection. The incidence
of listeriosis among other age ranges has remained unchanged and the incidence
in pregnant women has been stable since the early 1990s. This presentation
will explore the possible reasons for the increase in the over 60s focussing on several
strands of work that are being undertaken to address this. Firstly in 2007 the UK
Advisory Committee on the Microbiological Safety of Food (ACMSF) considered
that the change in the epidemiology seen in the UK was most likely linked to social
factors rather than changes occurring in the microorganism and referred the issue of
listeriosis in the elderly to its ad hoc group on vulnerable groups for further consideration.
The presentation will highlight some of the group’s findings which are available
in a report (www.food.gov.uk/multimedia/pdfs/committee/acmsflisteria.pdf ).
Amongst their recommendations they recognised the importance of food consumption
and food handling behaviours in the over 60s including those in vulnerable
groups. One of the report’s recommendations was for this to be considered by the
Food Standards Agency’s (FSA) Social Science Research Committee (SSRC). The
presentation will highlight findings from the SSRC’s report which showed that very
little is known about food storage and handling practices of over 60s in the home.
Studies suggest that older people handle food differently from younger people although
the reason for this difference is unclear. Specific differences reported included
poor refrigeration and defrosting practices; differences in cooling, storage
and reheating of leftovers; variable adherence to ‘use-by’ and ‘best-before’ dates; and
differences in personal and domestic hygiene such as hand-washing and cleaning of
kitchen surfaces. The report noted that there is relatively little evidence regarding
the current levels of food hygiene knowledge among those aged 60 years and over
and no information on whether the level of knowledge differs with generations or
has changed as people age. Emphasising the need for correct storage and handling of
food in the home particularly by those over 60s was a key theme of Food Safety week
in the UK in 2009 and the presentation will illustrate the approach that was taken by
the FSA in this campaign. The presentation will conclude by highlighting some of
the gaps in our knowledge and the SSRC recommendations concerning research.
* Plenary Supported by FCT
Consumer perceptions, behaviour and microbial food safety;
implications for Listeria control
Frewer, L. J.*
Wageningen University, The Netherlands
In order to understand fully the problem of food safety linked to the occurrence of listeriosis,
it is important to consider how consumers respond to food safety issues, in terms
of their psychology and how this determines their behaviour, for example in relation to
food preparation behaviour. An important research objective relates to consumer activities
after food purchase. Risk perception determines how consumers react to different
types of risks. Very generally, risks which are perceived to be unnatural in origin
and involuntarily imposed on the individual who is exposed to them are perceived to be
more threatening. Microbiological risks are perceived to be “naturally occurring” and,
in the case of food safety risks, highly controllable, and so are not a focus of consumer
concern. In addition, consumers tend to exhibit an “optimistic bias” in relation to microbiological
food risks, which means that food safety information tends to be perceived
as applying to more vulnerable, consumers in the population. Research suggests
that consumers are reasonably knowledgeable about safe food preparation practices,
but that this knowledge is not always applied in practice. Food preparation tends to be a
habitual behaviour, which is difficult to change. Introducing food safety messages during
food preparation (for example, in recipe development) tends to activate existing
food safety knowledge, as does the inclusion of materials designed to elicit affective responses
to food safety issues, for example disgust. Some groups within the population
are more vulnerable than others, and targeting risk communication messages to the
needs of these groups is particularly relevant. It is argued that, whilst consumer observation
studies, combined with modelling of critical control points in food preparation,
might indicate the riskiest behaviours in terms of Listeria, this information should be
combined with activation of general food safety knowledge if an effective approach to
reducing the incidence of food borne disease is to be developed.
* Plenary Supported by FCT
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ORAL PRESENTATIONS

AREA // A // Biology of Listeria monocytogenes
ORAL PRESENTATIONS // O / 01–13
The cold shock associated proteins (Csps) promote tolerance of different
environmental stresses and host cell invasion of Listeria monocytogenes
Tasara, T. 1, Klumpp, J. 2, Loessner, M. J. 2 and Stephan, R. 1
1. Institute of Food Safety, University of Zurich, Switzerland
2. Institute of Food, Nutrition and Health, ETH Zurich, Switzerland
The food-borne pathogen Listeria monocytogenes has to withstand various stress
conditions in order to survive and proliferate on foods and within different types
of host cells. The bacterial cold shock family proteins (Csps) consist of small,
highly conserved nucleic acid-binding proteins, and are assumed to have an influence
on the expression of various microbial genes. In addition to cold adaptation
functions, some prokaryotic Csp proteins are also involved in promotion of other
cell processes during normal bacterial growth, as well as in adaptation to nutrient
starvation and stationary growth phase stresses. The possible functional contribution
of L. monocytogenes CspA, CspB and CspD proteins were investigated
under food-related environmental stress, and during host cell invasion. We show
that the three L. monocytogenes Csp components, although highly homologous,
provide distinct cellular functions during cold, osmotic and oxidative stress adaptation
as well as host cell invasion processes. All three csp genes are constitutively
expressed but are dispensable for viability and growth of this organism at optimal
temperature. However, a hierarchy in Csp functional importance during cold
(CspA > CspD > CspB) and osmotic (CspD > CspA / CspB) stress adaptation is observed.
With respect to double (DcspBD) and triple (DcspABD) csp deletion mutants,
we also observe reduced survival of oxidative stress and a reduced ability to
invade Caco-2 and J744A.1 murine macrophages cells. Overall, our data indicate
important functional roles of L. monocytogenes Csp proteins in promotion of cellular
functions which facilitate environmental stress resistance and host cell invasion
processes of this food-borne pathogen.
Different levels of flagellin detected during growth
of Listeria monocytogenes strains at low temperature
Cabrita, P. 1,2,3*, Batista1, S., Moes, S. 4, Jenö, P. 4, Trigo, M. J. 3, Boavida Ferreira, R. 1,2
and Brito, L. 1
1. CBAA/ Departamento de Botânica e Engenharia Biológica, Instituto Superior de Agronomia,
Technical University of Lisbon, Lisbon, Portugal
2. Instituto de Tecnologia Química e Biológica, New University of Lisbon, Oeiras, Portugal
3. Instituto Nacional dos Recursos Biológicos, IP, Oeiras, Portugal
4. Department of Biochemistry, Biozentrum of the University of Basel, Basel, Switzerland
Listeria monocytogenes can tolerate a wide range of environmental stresses.
Growth at low temperatures is a stress that L. monocytogenes often has to face
since refrigeration is present throughout the food chain. This study aimed to investigate
whether unique or common proteins are up- or down-regulated under
nutritional stress conditions and low temperature. To achieve this, a simplified
methodology to analyse the extracellular protein profiles of four L. monocytogenes
strains was used. These strains, belonging to four different serovars, were selected
according to differences in virulence. Cultures were incubated in minimal medium
(Modified Welshimer Broth) at 10 °C. Proteins present in the supernatants of the
cell cultures, in late exponential phase, were precipitated and separated by SDS-
PAGE, using equivalent amounts of total secreted proteins. For each bacterial
strain, at least three independent culture assays were set. The most abundant
polypeptide bands and those suggesting the widest range in differential expression
among strains were identified by ESI LC / MS-MS. The p60 virulence protein,
one of the major proteins previously detected at 37 °C, was still detected in
relatively high amounts at 10 °C in all strains. The virulence proteins listeriolysin
O and internalin C expressed at 37 °C, were not detected at 10 °C in all strains, confirming
previous findings. In serovar 1/2a strain, flagellin was the major protein
identified. Two other strains, from the rare serovares 4c and 4d/4e, showed lower
levels of flagellin whereas for the serovar 4b strain no flagellin was detected. Flagella
have been shown to act as mediators in bacteria attachment to stainless steel
surfaces. Serogroup 1/2a has been reported as prevalent among food isolates.
Therefore, the different levels of flagellin detected may be related with the ability
of some strains to colonize food contact surfaces at refrigeration temperatures.
This hypothesis will be further investigated.
* Participation Supported by IUFoST
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AREA // A // Biology of Listeria monocytogenes
ORAL PRESENTATIONS // O / 01–13
Phenotypic and corresponding transcriptomic responses of Listeria
monocytogenes strains in the presence of unprotonated organic acids
Lee Chang, K. J., Pinfold, T., Koshy, A. and Bowman, J. P.
University of Tasmania, Australia
Sodium diacetate-resistant L. monocytogenes food and clinical isolates delineated
through a culture-based screening process were found to possess significantly better
survival levels following challenge to pH 2.4 acid challenges compared to various
reference strains. Sodium diacetate was found to directly and substantially
improve survival and was synergistic with acid tolerance resultant from the shift to
the stationary growth phase. The resistant strains had comparatively lower intracellular
levels of acetate and K + ions that corresponded to lower transmembrane
delta pH values. Another observed feature was that when grown in the presence of
sodium diacatete cell wall stability was enhanced as revealed by lysis assays, utilising
bead-beating and mutanolysin, suggesting acetate induces various cell wall
modifications that may also influence diffusion of unprotonated organic acid into
cells. Transcriptomic analysis of pH 5.0-habituated sodium diacetate-resistant
strain FW04/0025 compared with reference strain EGD that were grown with
21 mM (8 mM unprotonated) sodium diacetate at pH 5.0 indicated substantial variation
in genetic responses with EGD much more reactionary to the presence of
mineral acid. These differences were reflected in regulon-level gene expression
trends with EGD strongly activating and repressing the SigB- and CodY regulons,
respectively, while in FW04/0025 the response of these regulons were muted. The
transcriptome of FW04/0025 only becomes more congruent with that of EGD
when in the presence of sodium diacetate, though several distinct genetic expression
differences still occur. Gene expression trends deriving from exposure to
sodium diacetate suggest extensive cell wall and membrane modification, alterations
to branched-chain amino acid/fatty acid metabolism/biosynthesis, induction
of an SOS-like DNA repair and thioredoxin-mediated antioxidant responses
occurs, though strain-level specific responses are quite divergent. Corresponding
proteomic analyses are underway to further characterize responses to food preservative
organic acids by L. monocytogenes.
Internalin profiling, multilocus sequence typing
and virulence assesments suggest evolutionary history
of the Listeria monocytogenes-Listeria innocua clade
Chen, J. and Fang, W.
Zhejiang University, China
The morphological, ecological, biochemical and genetic resemblance, and the clear
difference of virulence between L. monocytogenes and L. innocua make this bacterial
clade attractive as models to examine the evolution of pathogenicity. This
study was attempted to examine the population structure of L. monocytogenes and
L. innocua, and further to investigate the microevolution in this clade via profiling
of 37 internalin genes, MLST analysis of gyrB-sigB-dapE-hisJ-ribC-purM-gap-tufbetL
gene cluster, and detection of 17 virulence genes, together with in vitro and in
vivo virulence assessments. Results indicate that L. monocytogenes comprises three
recognized lineages I, II and III, including a set of lineage III strains displaying
strong phospholipase activity and subdued virulence. While resembling L. monocytogenes
in having a nearly identical Listeria pathogenicity island I, and inlA and
inlB, these nonpathogenic L. monocytogenes strains harbor notably altered prfA,
actA and plcB, and share many similar internalin gene deletions with L. innocua,
e.g., inlJ, inlC, inlI and inlGHE. On the other hand, L. innocua represents a young
species descending from L. monocytogenes, which comprises four subgroups: two
major subgroups I and II, and one atypical subgroup IV exhibiting the least genetic
distance to L. monocytogenes. All L. innocua strains lack 17 virulence genes
found in L. monocytogenes, except for subgroup IV strains harboring inlJ, and are
nonpathogenic to mice. As shown by the estimation of the time to the most recent
common ancestor, L. monocytogene lineages I and II appeared at approximately
the same time, and this is also the case with L. innocua subgroups I and II. The
nonpathogenic L. monocytogenes strains and L. innocua subgroups IV constitute
the possible evolutionary intermediates between L. monocytogenes and L. innocua.
The evolutionary history in the L. monocytogenes-L. innocua clade represents a
rare example of evolution towards reduced virulence of pathogens.

The SOS response of Listeria monocytogenes is involved
in stress resistance, mutagenesis, and biofilm formation
van der Veen, S. 1,2 and Abee, T. 1,2
AREA // A // Biology of Listeria monocytogenes
ORAL PRESENTATIONS // O / 01–13
1. Top Institute Food and Nutrition (TIFN), Wageningen, The Netherlands
2. Laboratory of Food Microbiology, Wageningen University and Research Centre, Wageningen,
The Netherlands
The food-borne pathogen Listeria monocytogenes is widely distributed in the environment.
As a consequence, raw materials used by the food industry could introduce
L. monocytogenes to food processing facilities. L. monocytogenes has
evolved various strategies and networks to survive and adapt to changing conditions
e.g. during food processing. One of the stress response mechanisms we recently
identified in L. monocytogenes is the SOS response.
The SOS response is a conserved inducible pathway that is involved in DNA repair
and restart of stalled replication forks. We identified the SOS response regulon of L.
monocytogenes and showed that it is important for stress resistance and adaptive
mutagenesis. In the present study, we investigated the role of the SOS response in
L. monocytogenes biofilm formation. L. monocytogenes static biofilms on polystyrene
and glass consists of a homogeneous layer, while on stainless steel L. monocytogenes
biofilms consist of single attached cells or microcolonies. Static biofilms
contain the small rod-shaped morphology, which is very similar to the morphology
of planktonic cells. However, L. monocytogenes continuous flow biofilms consist of
ball-shaped microcolonies, which are surrounded by a dense network of knitted
chains composed of elongated cells. We showed that continuous flow biofilm formation
and not static biofilm formation is dependent on the SOS response. Using
Q-PCR analysis, promoter reporters, and SOS response mutants, we showed that
the SOS response is activated during knitted-chain biofilm formation and that deletion
of its regulon member yneA, which is involved in cell elongation during SOS response
activation, results in diminished biofilm formation in continuous flow conditions.
Furthermore, we demonstrated that activation of the SOS response during
continuous flow biofilm formation induced mutagenesis: wild-type biofilms
showed considerably higher rifampicin resistant fractions than ∆recA biofilms or
wild-type planktonic cultures. Our results show that the SOS response of L. monocytogenes
is important for stress resistance, adaptive mutagenesis, and continuous
flow biofilm formation, and may therefore contribute to the survival and persistence
of this pathogen in food processing environments.
Clonal diversity of Listeria monocytogenes, a worldwide perspective
Chenal-Francisque, V. 1, Lopez, J. 1, Cantinelli, T. 1, Caro, V. 2, Tran, C. 2, Leclerq, A. 1,
Lecuit, M. 1 and Brisse, S. 2
1. Institut Pasteur, National Reference Center and WHO Collaborating Centre for LISTERIA, Paris, France
2. Institut Pasteur, Genotyping of Pathogens and Public Health Platform, Paris, France
Listeria monocytogenes is a foodborne pathogen that can cause listeriosis, a severe
invasive disease in human with a high fatality rate. L. monocytogenes is widespread
in nature. Molecular typing methods have grouped L. monocytogenes into two
major genetic lineages (lineages I and II) and an additional minor lineage (lineage
III). They differ according to virulence and ecological origin. We have developed a
DNA sequencing-based subtyping method, multilocus sequence typing (MLST) to
examine the epidemiology and population genetics of L. monocytogenes. In the
present study, we have undertaken the first spatiotemporal analysis of L. monocytogenes
biodiversity by sequencing internal portions of seven housekeeping genes
in 300 strains isolated from the six continents. We selected a set of 300 unrelated
strains of L. monocytogenes collected from 41 countries and 6 continents isolated
between 1935 and 2009 from different sources. MLST data based on seven housekeeping
genes (3,288 nucleotides) were obtained as described previously (Ragon et
al., PLoS Pathogens (2008)). The results show a pattern of biodiversity similar to
the one we had initially reported in France. Indeed, the population structure is
similar to that obtained on 360 strains isolated principally from France by Ragon
et al. Sequences and allelic profiles are available on the Internet-accessible database
(www.pasteur.fr/mlst). L. monocytogenes appears to exhibit a homogeneous
clonal diversity across continents, indicating a high dispersal rate at a global scale.
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AREA // A // Biology of Listeria monocytogenes
ORAL PRESENTATIONS // O / 01–13
Life without a cell wall: Listeria monocytogenes L-form cells feature
a unique mode of division
Briers, Y., Dell’Era, S., Schuppler, M. and Loessner, M. J.
Institute of Food, Nutrition and Health, ETH Zurich, Switzerland
Cell wall-deficient bacteria referred to as L-forms have lost the ability to maintain
or build a rigid peptidoglycan envelope. Although L-forms had been studied
for decades and many reports exist on their morphological, serological and biochemical
properties, very little was known about the basic cell biology and molecular
mechanisms underlying the transformation process. Of particular interest is
the question how L-form bacteria are able to proliferate in the absence of a mature
cell wall. We have investigated the biology of stable, non-reverting Listeria monocytogenes
L-form cells (native and GFP labelled). Transmission electron microscopy
demonstrated that L-form cells are devoid of the typical thick Listeria
type cell wall small, and form small protoplast-like vesicles as well as multi-nucleated
macro-cells, surrounded only by a cytoplasmic membrane. They lack peptidoglycan-bound
proteins such as Internalin A, whereas membrane-anchored
proteins such as Internalin B are still present. Monitoring L-form growth by timelapse
confocal laser scanning microscopy revealed the development and maturation
of vesicles within maternal L-form cells. We propose a novel model for growth
and division of L-form bacteria, which may explain their ability to multiply in the
absence of a rigid cell wall. Furthermore, transcriptome analysis of parental and Lform
L. monocytogenes was performed using whole genome hybridization arrays.
Compared to parental bacteria, L-forms feature downregulated metabolic functions
correlating with the dramatic shift in surface to volume ratio, whereas upregulation
of stress genes reflects the difficulties in adapting to this unusual, cellwall
deficient lifestyle. We also observed that L-form cells taken up into
macrophages are not killed but seem able to survive for prolonged periods of at
least 48 hours. In conclusion, we show that L. monocytogenes L-forms (i) can arise
and survive in the environment, (ii) are able to multiply and divide, and (iii) show
intracellular survival in macrophages.
Pangenomic analysis of Listeria monocytogenes
Deng, X. 1, Phillippy, A. M. 2, Li, Z. 1, Salzberg, S. L. 2, Tortorello, M. L. 3 and Zhang, W. 1
1. National Center for Food Safety and Technology, Illinois Institute of Technology, Summit, USA
2. Center for Bioinformatics and Computational Biology, University of Maryland, College Park, USA
3. National Center for Food Safety and Technology, Food and Drug Administration, Summit, USA
Listeria monocytogenes is well known for its adaptability to diverse environment
and host niches and its high fatality rate among infected immunocompromised
populations. Three genetic lineages have been identified in this species. Strains
of genetic lineages I and II account for >90 % of human infections in the United
States, whereas strains from genetic lineage III are rarely implicated in human
infections for unclear reasons. Here we compare the genomes of 26 L. monocytogenes
strains representing the three lineages based on both in silico comparative
genomic analysis and high-density, pan-genomic DNA array hybridizations. We
uncover 86 genes and 8 small regulatory RNAs that likely make L. monocytogenes
lineages differ in carbohydrate utilization and stress resistance during their residence
in natural habitats and passage through the host gastrointestinal tract. We
also identify 2,330 to 2,456 core genes in the listerial pan-genome that define this
species and assess the impact of lysogenic bacteriophages on genomic diversification.
Phylogenomic reconstructions based on 3,560 homologous groups suggest
a polyphyletic population infrastructure and gradual loss of genes as this saprophytic
species diversified into a rare and probably defective lineage.

AREA // A // Biology of Listeria monocytogenes
ORAL PRESENTATIONS // O / 01–13
Evidence for an antiporter-independent glutamate decarboxylase
(GAD) system in Listeria monocytogenes: Influence of growth media
on GAD system activity
Karatzas, K.-A., Brennan, O., Heavin, S. and O’Byrne, C. P.
SFI, Ireland
The GAD system plays an important role in survival of Listeria monocytogenes
under acidic conditions (eg. acidic foods) and in virulence (eg. survival in stomach).
The GAD system imports extracellular L-glutamate (Glu) e through an antiporter,
converts it to γ-aminobutyric acid (GABA) resulting in the consumption
of an intracellular proton and thus the increase of the intracellular pH. We show
for the first time that (Glu) e added in Defined Medium (DM) is not used by the
GAD system and therefore it cannot increase the ability of this bacterium to grow
in mild acidic conditions, neither does it enhance acid resistance at lethal pH values
as it does in BHI. The rate of GABA export was monitored in BHI at various pH
values showing that it initiates at pH 4.6 and the rate increases as pH values decrease.
We also demonstrate that there are activators of the antiporter-based GAD
system in BHI, while its inactivity in DM is not due to the presence of inhibitors in
this medium. Activation was at the transcription level with the expression of
gadD2T2 being more than 100-fold higher in BHI than in DM where levels of
gadD2 were undetectable. Furthermore we demonstrated for first time that in
both acidified DM and BHI, L. monocytogenes accumulates high levels of intracellular
GABA (GABA) i (> 42 mM) and despite the slower rate of accumulation in DM
the final levels were identical in both media. Since DM does not contain any (Glu) e
we have shown for first time that this bacterium converts (Glu) i to (GABA) i , which
is not exported and thus is stored intracellularly. This suggests an alternative
mechanism of acid resistance based on the GAD system that circumvents the antiporter-based
mechanism. We suggest that the (GABA) i accumulation might
buffer the intracellular pH and thereby contribute in survival under extreme acidic
conditions.
Role of Listeria monocytogenes tyrosine phosphatases in conferring
listeriophage resistance
Paz, R.-N. 1, Eugster, M. R. 2, Zeiman, E. 1, Loessner, M. J. 2 and Calendar, R. 3
1. Hadassah Hebrew University Medical Center, Israel
2. Institute of Food, Nutrition and Health, ETH Zurich, Switzerland
3. Department of Molecular and Cell Biology, University of California, Berkeley, USA
Protein tyrosine phosphatase (PTP)-like proteins exist in many bacteria and are
segregated into 2 major groups: Low molecular weight, and conventional. These
PTP are suggested to be involved in many aspects of bacterial physiology including
stress response, DNA binding proteins, virulence and capsule/cell wall production.
By annotation Listeria monocytogenes (LM) possesses 2 potential low molecular
weight and 2 conventional PTPs. Although no tyrosine kinases were identified
yet in LM, using Immunoprecipitation (IP) on total cell lysate and MS plus MS/MS
on the IP products we have identified at least 10 tyrosine phosphorylated proteins.
These proteins vary in their physiological function and were associated with carbohydrate
metabolism, DNA and RNA binding, processing and transcription and
transport. Using LM WT strain 10403S, we have created an in-frame deletion mutant
lacking all 4 PTPs, as well as 4 additional complemented strains harboring
each of the PTPs. No major physiological differences were observed between the
WT and the mutant lacking all 4 PTPs. However, the deletion mutant strain was
found to be resistant to listeriophages A511 and P35, and sensitive to other listeriophages
such as U153 and A118. This phage resistance was attributed to reduced
attachment to the cell wall. Additionally, the mutant lacking all PTPs was found to
lack N-acetylglucosamine in its teichoic acid. Phage sensitivity was rescued in a
complemented strain harboring a low molecular weight PTP (LMO2540). We also
found that attachment of the phages is partially restored by the same PTP and by
one with conventional weight PTP (LMO1800). Thus, it seems that PTPs probably
affect many processes in LM, but mostly resistance to listeriophages.
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AREA // A // Biology of Listeria monocytogenes
ORAL PRESENTATIONS // O / 01–13
Thiolomics – the thiol: disulfide redox metabolism
of Listeria monocytogenes
Ondrusch, N. 1, Gopal, S. 2, Fuss, A. 1, Hagen, N. 1, Stoll, R. 1, Aharonowitz, Y. 3 and Kreft, J. 1
1. University of Würzburg, Biocenter, Germany
2. Dept. of Microbiology , University of Mysore, India
3. Dept. of Molecular Microbiology and Biotechnology, Tel Aviv University, Israel
The thiol: disulfide redox metabolism (TDRM), found in all living cells, constitutes
a multi-component network. It counteracts oxidative stress, serves to maintain
the proper intracellular redox potential, assists in protein folding, is essential
for the formation of deoxyribonucleotides for DNA synthesis and helps to repair
damaged proteins. The best-characterized biological thiols are the tripeptide glutathione
(GSH) and the small proteins of the thioredoxin (Trx) and glutaredoxin
(Grx) family. Oxidized GSH and Trx are recycled by cognate reductases (GSH reductase
– Gor/GshR; thioredoxin reductase – TrxB). A considerable number of
genes/gene products putatively involved in the TDRM of L. monocytogenes EGDe
has been identified in the genome sequence. It comprises the unique fused gene
gshF (GSH synthetase), grx, two putative gor/gshR, six members of the thioredoxin
family, trxB, class I and class III ribonucleotide reductases (nrdAB and
nrdDG), gpo/gpx, prx, tpx, ohrA/R (detoxification of organic hydroperoxides), msrA
(methionine sulfoxide reductase ) and several regulators, e.g. perR (peroxide regulon),
spx & rex (redox-sensing regulators), fur, zur & mntR (metal uptake), nrdR
and the redox-sensitive chaperone hsp33. We investigated the regulation and function
of these genes/gene products in the TDRM in particular and in the physiology
of L. monocytogenes in general, also with respect to infectivity and virulence, by the
following approach: i) construction of selected mutants, ii) study of their multiplication
in vitro and in vivo, and iii) genome-wide transcription profiling of wild
type and mutants under several in vitro conditions (oxidative and disulfide stress)
and in eukaryotic host cell models. A major result was that mutants defective in the
glutathione/glutaredoxin system showed extensive changes in their transcription
profiles. Affected were virulence genes, genes for regulators, transporters, stress
response factors and also for metabolic enzymes. These results emphasize the central
role of the TDRM, their impact on cellular processes and pathogen-host interaction
will be discussed.
RNA-structures acting at a distance
Johansson, J.
Umeå University, Sweden
Riboswitches are RNA-elements acting in cis, controlling expression of their
downstream genes through a metabolite-induced alteration of their secondary
structure. Here, we demonstrate that an S-adenosylmethionine (SAM) riboswitch,
SreA, in Listeria monocytogenes can also function in trans, and act as a non-coding
RNA. We show that SreA controls expression of the virulence regulator PrfA by
binding to the 5´-untranslated region of its mRNA in a temperature-dependent
manner. Absence of the SAM riboswitch increases the level of PrfA. Thus, the impact
of the SAM riboswitch on PrfA highlights a link between virulence and the
nutritional status of the bacterium. Together, our results describe a novel role for
riboswitches and a new class of regulatory non-coding RNAs in bacteria.

AREA // A // Biology of Listeria monocytogenes
ORAL PRESENTATIONS // O / 01–13
Deep RNA sequencing of Listeria monocytogenes reveals overlapping
and extensive stationary phase and sigma B-dependent transcriptomes,
including multiple highly transcribed noncoding RNAs
Oliver, H. F. 1, Orsi, R. H. 1, Ponnala, L. 1, Keich, U. 2, Wang, W. 1, Sun, Q. 1, Cartinhour, S. 3,
Filiatrault, M. J. 3, Wiedmann, M. 1 and Boor, K. J. 1
1. Cornell University, USA
2. University of Sydney, Australia
3. United States Department of Agriculture-Agricultural Research Service, Robert W. Holley Center for
Agriculture and Health, USA
Identification of specific genes and gene expression patterns important for Listeria
monocytogenes survival, transmission and pathogenesis is critically needed to
enable development of more effective control strategies. The stationary phase
stress response transcriptome, which includes many sigma B-dependent genes,
was defined in L. monocytogenes using RNA sequencing (RNA-Seq) with the Illumina
Genome Analyzer. Specifically, transcriptomes were compared between stationary
phase cells of L. monocytogenes 10403S and an otherwise isogenic ∆sigB
mutant, which does not express the alternative sigma factor σ B, a major regulator
of genes contributing to stress response, including stresses encountered upon
entry into stationary phase. Overall, 83 % of all L. monocytogenes genes were transcribed
in stationary phase cells; 42 % of currently annotated L. monocytogenes
genes showed medium to high transcript levels under these conditions. A total of
96 genes had significantly higher transcript levels in 10403S than in ∆sigB, indicating
σ B-dependent transcription of these genes. RNA-Seq analyses indicate that
a total of 67 noncoding RNA molecules (ncRNAs) are transcribed in stationary
phase L. monocytogenes, including 7 previously unrecognized putative ncRNAs.
Application of a dynamically trained Hidden Markov Model, in combination with
RNA-Seq data, identified 65 putative σ B promoters upstream of 82 of the 96 σ Bdependent
genes and upstream of the one σ B-dependent ncRNA. The RNA-Seq
data also enabled annotation of putative operons as well as visualization of 5’- and
3’-UTR regions. The results from these studies provide powerful evidence that
RNA-Seq data combined with appropriate bioinformatics tools allow quantitative
characterization of L. monocytogenes transcriptomes, thus providing exciting new
strategies for exploring transcriptional regulatory networks.
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AREA // B // Listeria monocytogenes as a human and animal pathogen
ORAL PRESENTATIONS // O / 14–23
Probiotics reduce Listeria monocytogenes-induced tissue invasion and
stillbirths in pregnant guinea pigs
Smith, M. A., Agyekum, K. and Williams, D.
Environmental Health Science Department, University of Geórgia, USA
One-third of listeriosis cases are pregnancy-related and can lead to miscarriage
or stillbirth, premature delivery, or infection of the newborn. Recently we have
published a risk assessment based on dose response data from nonhuman primates
and guinea pigs. Both animal models estimate LD 50 s of approximately 10 7 L.
monocytogenes cfu similar to the FAO/ WHO estimated human LD 50 of 1.9 x 10 6
cfu based on outbreak data. The similarities between the dose response curves
and LD 50 s suggest these animal models are appropriate for testing therapies and
preventive strategies applicable to humans. Recently, probiotics have been proposed
to protect hosts from pathogens introduced to the system through ingestion.
Our objective was to determine the efficacy of probiotics in yogurt in preventing
L. monocytogenes invasion and stillbirths. Using our pregnant guinea pig
model for listeriosis, 5 ml of yogurt containing Lactobacillus and Bifidobacterium
was administered orally to pregnant guinea pigs on gestation days (gd) 32–36. On
gd 35, guinea pigs were orally fed L. monocytogenes (10 9 cfu) in sterilized whipping
cream at four hrs after yogurt feeding. By gd 56 in those animals receiving only L.
monocytogenes, the pathogen was isolated from 100 %, 75 %, 64 %, 71 % and 71 %
of maternal livers and spleens, placentas, fetal livers and brains, respectively. However
in those that received yogurt and L. monocytogenes, the pathogen was isolated
from 56 %, 14 %, 17 %, 17 % and 22 % of maternal livers and spleens, placentas,
fetal livers and brains respectively. Also, 75 % of pregnant guinea pigs treated with
10 8 L. monocytogenes cfu have stillbirths compared to 14 % when treated with yogurt
and L. monocytogenes. These results present opportunities to develop treatment
and preventive therapies for listeriosis. In summary, consumption of yogurt
containing Bifidobacterium and Lactobacillus reduced the invasion and number
of stillbirths after L. monocytogenes exposure in pregnant guinea pigs.
Listeriolysin O favors Listeria monocytogenes growth in co-culture with
the ciliate Tetrahymena pyriformis
Ermolaeva, S. and Pushkareva, V.
Gamaleya Institute of Epidemiology and Microbiology, Russian Federation
Listeria monocytogenes was isolated from soil, water, sewage and sludge. We explored
the potential of L. monocytogenes major virulence factor Listeriolysin O
(LLO) to promote interactions between L. monocytogenes and the ubiquitous inhabitant
of natural ecosystems bacteriovorous free-living ciliate Tetrahymena pyriformis.
Axenic T. pyriformis and the following bacterial strains were used: wild
type L. monocytogenes strains EGDe, VIMVR081, VIMVW039, VIMHA034,
VIMVF870, EGDe derivative EGDe::Dhly and wild type L. innocua strain
NCTC11288. Experiments were performed in LB broth at 28 °C. Bacteria were
counted bacteriologically or by qPCR. T. pyriformis trophozoites and cysts were
counted using light microscopy. The hly gene was introduced into pTRKH2 vector,
and the same plasmid supplemented with prfA* gene was used to express LLO in L.
innocua. Guinea pigs were infected intraconjunctivally or per os with L. monocytogenes
culture or with T. pyriformis cysts infected L. monocytogenes. After 7 days
of co-culturing, wild type L. monocytogenes strains reduced trophozoite and increased
cyst concentrations up to 21.1 and 6.1 times, respectively. EGDe::Dhly
failed to cause mortality among protozoa and to trigger protozoan encystment. In
concordance, LLO deficiency deteriorated L. monocytogenes growth in the presence
of T. pyriformis. Replenishment of the hly gene in the mutant strain restored
toxicity towards protozoa. L. innocua transformed with the LLO-expressing plasmid
caused extensive mortality and encystment in ciliates. L. monocytogenes EGDe
entrapped in cysts caused infection in guinea pigs upon ocular and oral infection.
The L. monocytogenes virulence factor LLO promotes bacterial survival and is responsible
for L. monocytogenes toxicity for protozoa and induction of protozoan
encystment. Therefore, LLO activity might support bacterial survival in the natural
habitat outside of a host.

Atopy is a risk factor for listeriosis
Kawamoto, K., Matsubara, S., Da Silva, M. and Makino, S.-I.
Obihiro Univ. Agri. Vet. Med., Japan
AREA // B // Listeria monocytogenes as a human and animal pathogen
ORAL PRESENTATIONS // O / 14–23
Listeria monocytogenes is a Gram-positive bacterium that causes meningitis, bacteremia,
and febrile gastroenteritis. The outcome of listeriosis is dependent on
host factors such as age, pregnancy, and HIV infection, which influence host immunocompetency.
A Th-1 type cytokine IFN-gamma; plays an important role in
the innate immune response against intracellular bacterial pathogens. In contrast,
Th2-biased immune responses often associate with most atopic diseases. In this
study, we examined whether atopy affected the immune response to L. monocytogenes
infection. NC/Nga is a model mouse for human atopic dermatitis, which has
a genetic predisposition to develop atopic skin lesion. We compared the susceptibility
to listeriosis of NC/Nga mice with BALB/c and C57BL/6 mice. The course of
infection was characterized by monitoring survival of mice, and determination of
bacterial numbers in the organs. NC/Nga mice were highly susceptible to L. monocytogenes
infection: the 50 % lethal dose was significantly lower and the number of
bacteria in livers, spleens and brains were higher in NC/Nga than those for other
inbred mice. The increased permeability of blood-brain barrier was observed in
NC/Nga brains at day 3 post infection, but not in BALB/c and C57BL/6. As compared
to BALB/c and C57BL/6 mice, plasma IFN-gamma-levels of NC/Nga were
comparative. However, markedly increased IL-10 levels were detected in NC/Nga
plasma. Pretreatment with neutralizing antibodies to IL-10 partially protected
mice but retarded the clearance of bacteria. Yet the molecular mechanisms by
which the infection of L. monocytogenes induces overproduction of IL-10 in
NC/Nga mice remain unknown, our results suggest that the differential cytokine
production may at least partially underlie the higher susceptibility to L. monocytogenes
in NC/Nga mice. Considering the increased prevalence of atopic diseases,
an atopic phenotype may be a potential risk factor for listeriosis.
Cancer immunotherapy using novel Listeria monocytogenes-bacterial
vectors to target the vasculature of progressive tumors
Paterson, Y., Seavey, M. M., Maciag, P. C. and Sewell, D.
University of Pennsylvania, USA
For nearly 20 years our laboratory has been developing Listeria monocytogenes
(Lm) as a vaccine carrier to introduce tumor and viral antigens to the immune
system for the immunotherapy of cancer and as prophylactic vaccines for infectious
disease. Given the antigenic instability of tumor cells, we recently turned
our attention to the use of immunotherapy to destroy cells actively involved in
forming new blood vessels that support the growth and spread of cancer. We constructed
Lm expression systems that would target two central cell types involved
in angiogenesis – endothelial cells and pericytes. Two proteins are highly expressed
on each cell during active angiogenesis – Vascular Endothelial Growth
Factor Receptor-2 (VEGFR2) (endothelial cells) and High Molecular Weight
Melanoma Associated Antigen (HMWMAA) (pericytes). We selected fragments
from each molecule that included peptides predicted to bind to the HLA-A2 molecule.
We fused the genes encoding these fragments to the gene encoding the first
420 residues of Listeriolysin-O (LLO) and used Lm to deliver these fusion proteins.
Even though the immune system should be tolerant to these self-molecules,
both vaccines elicited potent anti-tumor CTL responses. Lm-LLO-VEGFR2 was
able to reduce tumor microvascular density (MVD), cause regression of established
breast tumors and protect against tumor re-challenge 100+ days post last
immunization. The Lm-LLO-HMWMAA vaccine also dramatically reduced MVD,
induced regression of established breast tumors, increased CD8+ T cell infiltration,
and, in addition, reduced the pericyte coverage of intra-tumoral blood vessels.
Interestingly anti-tumor efficacy was dependent on epitope spreading to the
tumor-associated antigen Her-2/neu. Neither immunotherapeutic interfered with
the generation of normal vasculature in wound healing or gestation. We are currently
testing these vaccines to impact the progression and metastasis of advanced
breast cancer, we hypothesize that reduced angiogenesis will prevent the formation
of distal metastases blunting cancer spread and improving overall survival.
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AREA // B // Listeria monocytogenes as a human and animal pathogen
ORAL PRESENTATIONS // O / 14–23
The intracellular carbon metabolism of Listeria monocytogenes
in comparison to that of other intracellular bacterial pathogens
replicating in mammalian host cells
Gotz, A. 1, Eylert, E. 2, Stoll, R. 1, Eisenreich, W. 2, and Goebel, W. 3
1. Biocenter-Microbiology, University of Würzburg, Germany
2. Institute of Biochemistry, LMU München, Germany
3. Max-von-Pettenkofer Institute, LMU München, Germany
Intracellular bacterial pathogens replicate either in the cytosol (e.g. Listeria monocytogenes,
Shigella spp. and the closely related enteroinvasive Escherichia coli
[EIEC]) or in specialized phagosomal compartments (e.g. Salmonella enterica
Serovar Typhimurium) of the infected host cells. Our knowledge on the metabolic
adaptation processes between intracellular pathogens and their host cells, allowing
their efficient intracellular growth, and on the influence of the intracellular
bacterial metabolism on the expression of those virulence genes, that are decisive
for the intracellular life cycle of the intracellular pathogens, is still rather limited.
We have carried out initial studies concerning these important questions with the
above mentioned intracellular bacteria. For this goal we constructed mutants impaired
in the uptake and/or catabolism of specific carbon sources and studied by
NMR- or MS-based 13C-isotopologue profiling their intracellular carbon metabolism
in comparison to the isogenic wild-type strains after infection of suitable
mammalian host cells. The results obtained show that the intracellular carbon
metabolism of L. monocytogenes differs significantly from that of the two other
pathogens. Whereas glucose, but not glucose-6-P is the major carbon substrate
for intracellular growth of EIEC and S. typhimurium, L. monocytogenes uses C 3 -
substrates (e.g. glycerol) as major and glucose-6-P as subsidiary carbon sources.
The reason for this difference is apparently the lack of high affinity glucose transporters
in L. monocytogenes. However, the intracellular C-metabolism of all three
pathogens in mammalian host cells is surprisingly flexible. Mutants defective in
the uptake of the preferential carbon source switch readily to alternative carbon
sources and the lower energy supply of the utilized secondary carbon sources
seems to be compensated by an increased uptake of anabolic monomers from the
host cells. As shown for L. monocytogenes the intracellular carbon metabolism appears
to be adapted to optimal expression of the virulence genes that are essential
for the intracellular life style.
LIMP2 links late phagosomal trafficking with the onset
of the innate immune response to Listeria monocytogenes:
a role in macrophage activation
Fernandez-Prieto, L., Carrasco-Marin, E., Madrazo-Toca, F., Rodriguez-Del Rio, E.,
Carranza-Cereceda, C. and Alvarez-Dominguez, C.
Immunology Departament, Hospital Santa Cruz de Liencres and Fundacion Marques de
Valdecilla – IFIMAV, Spain
The innate immune response to Listeria monocytogenes depends on phagosomal
bacterial degradation by macrophages. Here, we describe the role of LIMP2, a lysosomal
type III transmembrane glycoprotein and scavenger-like protein, in Listeria
phagocytosis. We show here that LIMP2 is not involved in bacterial recognition at
the cell surface but participates in the degradation of Listeria within phagosomes.
LIMP2 appears linked to Rab5a activation and controls the late-endosomal/lysosomal
fusion machinery. Importantly, LIMP2 deficient mice display a
macrophage-related defect in innate immunity. They produce less acute-phase
pro-inflammatory cytokines/chemokines, MCP-1, TNF-α and IL-6, but normal
levels of IL-12, IL-10 and IFN-γ and a 20-fold increase in susceptibility to Listeria
infection. This macrophage dysfunction results in a low listericidal potential, impaired
phago-lysosome transformation into antigen-processing compartments
and uncontrolled LM cytosolic growth that fails to induce normal levels of acutephase
pro-inflammatory cytokines. Therefore, the role of LIMP2 appears to be
connected to the activation of Listeria-primed macrophages through internal signals
linking the regulation of late trafficking with the onset of the innate Listeria
immune response.

AREA // B // Listeria monocytogenes as a human and animal pathogen
ORAL PRESENTATIONS // O / 14–23
The tetraspanin CD81 is required for entry of Listeria monocytogenes
in mammalian cells
Tham, T. N. 1,2,3, Gouin, E. 1,2,3, Rubinstein, E. 4,5, Boucheix, C. 4,5, Cossart, P. 1,2,3
and Pizarro-Cerdá, J. 1,2,3
1. Institut Pasteur, Unité des Interactions Bactéries-Cellules, Département de Biologie Cellulaire
et Infection, Paris, France
2. INSERM, U604, Paris, France
3. INRA, USC2020, Paris, France
4. Institut André Lwoff, Université Paris-Sud, Villejuif, France
5. INSERM, U602, Villejuif, France
Listeria monocytogenes is a facultative intracellular bacterial pathogen that invades
epithelial cells by subverting signaling cascades associated with its two main
cellular receptors, E-cadherin and Met. We recently identified the type II phosphatidylinositol
4-kinases (PI4KIIs) α and β as required for bacterial entry downstream
of Met. In this work we investigated whether tetraspanins CD9, CD63 and
CD81, which figure among the few described molecular partners of the PI4KIIα,
function as molecular adaptors recruiting the PI4KIIα to the bacterial entry site.
We observed by fluorescence microscopy that CD9, CD63 and CD81 are expressed
and detected at the cellular surface and also within intracellular compartments,
particularly in the case of CD63. In resting cells, colocalization between these
tetraspanins and the PI4KIIα is only detectable in restricted areas of the perinuclear
region. Upon infection with Listeria, endogenous CD9, CD63 and CD81 were
recruited at the bacterial entry site but did not colocalize strictly with endogenous
PI4KIIα. Live cell imaging confirmed that tetraspanins and the PI4KIIα do
not follow the same recruitment dynamics to the Listeria entry site. Depletion of
CD9, CD63 and CD81 levels by small interfering RNA (siRNA) demonstrated that
CD81 is required for bacterial internalization, identifying for the first time a role
for a member of the tetraspanin family in the entry of Listeria within target cells.
Moreover, depletion of CD81 inhibits recruitment of PI4KIIα to the bacterial entry
site but not that of the Met receptor, suggesting that this tetraspanin could act as
a membrane organizer required for the integrity of signaling events occurring at
Listeria entry sites.
Listeria innate immune evasion by peptidoglycan modification
Aubry, C.
Department of Cellular Biology and Interaction, Institut Pasteur, France
Peptidoglycan (PG) plays an important role in host-pathogen interaction because
PG is the site of anchoring of virulence factors and an important target for the innate
immune system. As reported previously, inactivation of pgdA, encoding a PG
N-deacetylase in Listeria monocytogenes, revealed a key role of PG modification
in virulence as survival of the mutant was severely impaired in mice (Boneca et
al., PNAS 2007). Deletion of the pgdA gene highly increased sensitivity to the bacteriolytic
activity of hen egg lysozyme in vitro. The pgdA mutant was rapidly destroyed
within phagosomes and induced a potent pro-inflammatory response by
macrophages. Interestingly, inactivation of the deacetylase induced a strong secretion
of IFN beta by infected macrophages, through a surprisingly and uncharacterized
TLR2-dependent pathway. We have now shown that TLR9 but not TLR4
contribute to this IFN beta secretion. The key adaptors and the new signaling
pathway leading to IFN beta production are under investigation. We found that
L. monocytogenes PG is also modified by a putative O-acetyltransferase (OatA) essential
for virulence. A Listeria oatA mutant shows an increased sensitivity to hen
egg lysozyme, bacteriocin and cell wall targeting antibiotics in vitro. Its growth is
impaired in macrophages, possibly contributing to immune escape. Modification
of PG is a highly efficient mechanism used by pathogenic Listeria to evade innate
host defenses.
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AREA // B // Listeria monocytogenes as a human and animal pathogen
ORAL PRESENTATIONS // O / 14–23
Constitutive activation of the central virulence transcriptional
regulator PrfA enhances Listeria monocytogenes pathogenesis but
reduces bacterial fitness outside of the host
Freitag, N. E. 1 and Bruno, J. C. 2
1. Department of Microbiology & Immunology, University of Illinois at Chicago, USA
2. Department of Global Health, University of Washington, USA
Listeria monocytogenes survives as a saprophyte in soil and decaying vegetation
while maintaining an ability to invade mammalian cells and cause serious disease.
Survival and replication of L. monocytogenes within mammalian hosts requires
the regulated synthesis and expression of multiple gene products that enable host
cell invasion, bacterial replication within the cytosol, and spread to adjacent cells.
The transcriptional regulator PrfA controls the expression of multiple bacterial
virulence factors and is required to facilitate the L. monocytogenes transition from
saprophyte to pathogen. PrfA has been shown to exist in both low and high activity
states, with the transition to high activity occurring within the cytosol of host cells.
A number of mutations within prfA have been described (prfA* mutations) that
serve to lock the protein into a high activity state. We examined the consequences
of constitutive PrfA activation on L. monocytogenes physiology and pathogenesis
by assessing the fitness of prfA* strains in a variety of in vitro and in vivo conditions.
prfA* strains exhibit a competitive advantage over wild strains in animal
models of infection, with ten-fold higher numbers of prfA* bacteria recovered from
the livers and spleens of infected mice. However, despite apparently normal
growth in broth culture, the prfA* mutants exhibited a fitness defect when grown
in the presence of wild type bacteria, and this fitness defect was exacerbated when
mixed cultures were placed under various stress conditions. Strains containing
prfA* mutations were also defective in their ability to adapt to a long term survival
phase following prolonged growth in broth culture. Taken together, these results
indicate that L. monocytogenes must maintain a critical balance of PrfA activity
to promote bacterial fitness both inside and outside of infected host cells.
The lvfH gene of Listeria monocytogenes encodes a novel virulence factor
highly activated during infection
Carvalho, F., Camejo, A., Ferreira, P., Sousa, S. and Cabanes, D.
IBMC – Instituto de Biologia Molecular e Celular, Group of Molecular Microbiology,
Universidade do Porto, Portugal
Listeria monocytogenes is a highly adaptable Gram-positive bacterium that can
induce potentially lethal listeriosis in immunocompromised human hosts. As a
facultative intracellular pathogen, it is able to invade and replicate inside different
eukaryotic cell types and, by cell-to-cell spread, disseminate infection. Such
processes are highly dependent upon expression of specialized proteins which
act as virulence factors on certain steps of the bacterial infectious cycle. Our recent
studies on the L. monocytogenes EGD-e transcription profile in infected
mice revealed genes that were highly activated throughout the infection timeline
and could be involved in virulence. Among these is lvfH, a serotype 1/2a-specific
gene that encodes a protein putatively involved in the L-rhamnose biosynthesis
pathway, a mechanism that provides substrate for the decoration of cell wall teichoic
acids. In order to confirm and characterize the role of this gene in L. monocytogenes
virulence, we generated an lvfH deletion mutant and investigated the
influence of this mutation in the Listeria infectious process. In vitro assays
showed that the mutant was unaffected in its host cell membrane-adhering
properties, but was significantly impaired in its ability to invade different mammalian
cell lines. Organs of mice intravenously infected with the lvfH mutant
displayed a significant decrease in bacterial load, as compared to animals infected
with wild type bacteria. This phenotype is unrelated with an intrinsic
growth defect of the mutant, as it presented growth profiles similar to the wild
type strain in broth and within murine macrophages. Animal organs infected
with an lvfH-complemented strain showed wild type-like infection levels, confirming
the specific requirement of LvfH for full virulence in this model.
Functional characterization of LvfH and analysis of the relation between teichoic
acid rhamnosylation and virulence could reveal a new role for teichoic acid
decoration in L. monocytogenes pathogenesis.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
ORAL PRESENTATIONS // O / 24–36
Human listeriosis due to Listeria ivanovii
Guillet, C. 1, Join-Lambert, O. 1, Le Monnier, A. 1, Leclercq, A. 2, Mamzer-Bruneel,
M. F. 1, Bielecka, M. K. 3, Scortti, M. 3, Disson, O. 2,4, Vazquez-Boland, J. 3, Lortholary, O. 1
and Lecuit, M. 1,2,4
1. Necker-Enfants Malades hospital, Paris Descartes University, Paris, France
2. Institut Pasteur, NRC and WHO-CC for Listeria, Paris, France
3. University of Edinburgh, Scotland, UK
4. Institut Pasteur, Microbes and host barriers Group, Inserm avenir, Paris, France
The genus Listeria contains two pathogenic species: Listeria monocytogenes (Lm)
that infects human and animals, and Listeria ivanovii subps. ivanovii (Lii), considered
a ruminant-specific pathogen. We describe here a case of gastroenteritis and
bacteremia due to Lii in a kidney transplant recipient. A 55-year-old man was referred
to our hospital with a three-week history of non-bloody diarrhoea, vomiting,
dehydratation, and low-grade fever. He underwent a renal transplantation for
chronic renal failure and was chronically infected with hepatitis C virus. Stool and
blood cultures allowed the isolation of Lii. Intravenous amoxicillin (6 g/day) and
gentamicin (6 mg/kg/day) treatment was associated with resolution of symptoms.
The 4 Lii isolates from this patient had indistinguishable ApaI and SmaI PFGE
patterns, phenotypic characters and classical resistance for antibiotics. These
human isolates were indistinguishable from prototypic ruminant strains based on
(i) the activation status of the central virulence gene regulator PrfA, (ii) the presence
of the L. ivanovii-specific pathogenicity island LIPI-2 by PCR mapping, and
(iii) invasion assays using Madin-Darby Bovine Kidney cells and huma, HeLa cells.
So, Lii is pathogenic for humans. As for Lm, Lii route of infection is foodborne. A
review of the literature identified 8 cases, mostly in immunosuppressed patients.
The rarity of human Lii infections probably reflects low exposure given the rare
occurrence of this species in nature compared to Lm.
Foodborne listeriosis in India: An update
Barbuddhe, S. B. 1*, Malik, S. V. S. 2, Ashok Kumar, J. 1, Kalorey, D. R. 3, Kurkure, N. V. 3,
Rawool, D. B. 4, Swain, B. K. 1, Korikanthimath, V. S. 1 and Chakraborty, T. 4
1. ICAR Research Complex for Goa, India
2. Division of Veterinary Public Health, Indian Veterinary Research Institute
3. Nagpur Veterinary College, India
4. Institute of Medical Microbiology, Justus Liebig University, Giessen, Germany
Listeria monocytogenes is a foodborne pathogen that can cause serious invasive
illness, mainly in certain well-defined high-risk groups, including elderly and immunocompromised
patients, pregnant women, newborns and infants. In India,
the pathogen has been isolated from humans, animals, a variety of foods including
milk and milk products, meat and meat products and vegetables. In India, studies
on molecular epidemiological aspects of L. monocytogenes are largely lacking.
Therefore, we do not know the genetic variability of strains isolated and their epidemic
potential. The genetic diversity of the Listeria strains from various sources
namely, milk and milk products (112), fish and seafood (47), wild life (7), poultry
meat (19), meat and processed meats (37), animal clinical cases (41) and human
clinical cases (21) isolated/collected from different places in the country have been
characterized biochemically and with in vitro assays before attempting for serotyping
and virulence gene profiles. Out of the strains characterized phenotypically,
195 strains have been serotyped using multiplex PCR and pulsed field gel electrophoresis.
The predominant serotype among Indian Listeria isolates is L. monocytogenes
4b. Significant variation among the isolates recovered from different
sources has been observed as evident by PFGE analysis. As many as 34 different
PFGE profiles have been observed. An electronic database for the characterized
strains has been created. This is an interactive web based database so that the data
can be exchanged between laboratories electronically. The molecular characterization
of Listeria isolates from India would help in better understanding of the
sources of infection and their risk assessment, routes of transmission of the infective
agent, influencing factors, clinical forms and virulence characteristic of
the pathogenic isolates of Listeria in order to diagnose the infection rapidly and reliably
but also in instituting the effective control measures.
* Participation Supported by Fundação do Oriente
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AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
ORAL PRESENTATIONS // O / 24–36
Human listeriosis and co-morbidities in England, 1999 to 2008:
quantifying the risk
Mook, P., Grant, K., O’Brien, S. J. and Gillespie, I.
Health Protection Agency, UK
Listeria monocytogenes causes a rare but severe food borne disease (listeriosis),
commonly affecting pregnant women, the elderly and the seriously ill. The epidemiology
of listeriosis in England and Wales changed between 2001 and 2008,
with more patients aged ≥ 60 years presenting with bacteraemia. We examined
the risks in this age group, and quantified the role of co-morbidities for listeriosis
in all age groups. Case-patients were resident in England between 1999 and 2008
and were reported to an enhanced national surveillance scheme by hospital microbiologists
using a clinical case questionnaire. We coded co-morbidities of non
pregnancy-related cases of L. monocytogenes infection according to ICD-10. These
data were compared with appropriate denominator data (Hospital Episode Statistics
finished consultant episodes), to calculate incidence rates per million consultations
(with appropriate 95 % confidence intervals). Between 1999 and 2008,
1412 non-pregnancy related cases of listeriosis were reported in England. We received
a clinical questionnaire for 81 % of cases (N=1141). Eighty-two percent
(N=934) had one or more underlying medical conditions and we recorded 1239
ICD-10 codes on co-morbidities from these 934 cases. The ≥ 60 years age group
comprised 76 % of all cases and 77 % of all co-morbidities. Overall, the highest comorbidity
rates were diseases of the liver (192.9 episodes per million [95 % CI:
150.9-242.9]), systemic connective tissue disorders (163.1 [108.4-235.7]), malignancies
of the lymphoid and haematopoietic tissue (137.5 [118.5-158.7]), alcoholism
(107.5 [80.8-140.3]), renal failure (103.5 [83.3-127.3]), diabetes (98.4 [76.8-124.1]),
hypertensive disease (71.7 [44.9-108.5]) and malignancies of the eye, brain & other
parts of CNS (66.3 [35.3-113.3]). We have highlighted several underlying conditions
not previously thought to be strongly associated with listeriosis. The extent
to which these co-morbidities are correlated with each other requires further investigation
to enable much better, targeted prevention.
Risk factors for death in Listeria monocytogenes infection, England and
Wales, 1990 to 2008
Mook, P., Grant, K. and Gillespie, I.
Health Protection Agency, UK
Listeriosis, caused by the Gram positive bacterium Listeria monocytogenes, is a
rare but severe food borne disease. The elderly, pregnant and the seriously ill are
most often affected, with high mortality rates in all patient groups. The epidemiology
of listeriosis in England & Wales has changed in recent years, with an average
of 179 cases reported annually from 2000 to 2008 compared with 110 cases on average
between 1990 and 1999. Much of the increase has occurred in patients > 59
years presenting with bacteraemia but without central nervous system involvement.
To examine factors influencing L. monocytogenes mortality, non-pregnancy
associated cases resident in England & Wales reported to national surveillance
between 1990 and 2008 (N = 1832) were analysed further. The overall mortality
rate for this period was 39 % (N = 719). Univariable and subsequent multivariable
analyses were performed and the role of key factors (season, age, underlying conditions
and treatment) in this outcome investigated. Our initial findings suggest
that, for a disease which disproportionately affects the elderly and the infirm, mortality
is greatest in older patients and in those with underlying conditions. Underlying
conditions themselves (particularly alcoholism and malignancies of the
lymphatic/haematopoietic system and the breast) appear to be more important
than the treatment they were receiving for an underlying condition, even when
the effect of age is considered. Given that listeriosis is largely a food-borne disease,
and therefore preventable, it would seem appropriate to actively target high
risk groups with specific advice on what foods to avoid in order to minimise the
risk of listeriosis.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
ORAL PRESENTATIONS // O / 24–36
A discrete event model to track Listeria monocytogenes
in the retail environment
Pouillot, R. 1 and Gallagher, D. 2
1. CFSAN/FDA, USA
2. Va Tech, USA
A recent comparative Listeria monocytogenes (Lm) risk assessment predicts that of
the listeriosis cases attributed to deli meat, a majority are associated with deli
meat sliced and packaged at retail. There is a need to: 1) identify potential sources
and practices that contribute to Lm contamination of food at the retail level; and 2)
identify retail practices that would reduce or eliminate Lm contamination of
ready-to-eat foods prepared and sold to consumers. Within the scope of an interagency
U.S. Food and Drug Administration (FDA) and U.S. Department of Agriculture/Food
Safety and Inspection Service (USDA/FSIS) risk assessment of Lm at
retail, a dynamic discrete-event model that tracks Lm cells at various locations
over time in a retail setting was developed. This model considers i) the transfer of
bacteria from some defined compartment to others (unsliced products, sliced
products, food contact surfaces, non food contact surfaces, slicer, gloves, hands,
etc.); ii) the bacterial inactivation through cleaning and disinfection; and iii) the
bacterial growth according to the physic and chemical environment (temperature,
pH, water activity, presence of growth inhibitors). Sequences of events were derived
using specifically acquired data from an observational study of food handling
practices in retail deli departments. Additional data on the transmission of
Lm in the retail environment is being collected through laboratory studies. Moreover,
this risk assessment is designed to evaluate the relative effectiveness of retail
Lm control measures along with other risk management scenarios. The major uncertainty
is in the potential existence of niches and their interaction with the retail
environment. This presentation will describe this model, provide an illustration
using current data and highlight the data gaps that mainly influence the outputs.
Clinical and epidemiological aspects of listeriosis in Israel
Hershko-Klement, A. 1, Eliav, H. 2, Valinsky, L. 3, Schechner, V. 4, Braun, E. 5, Paitan, Y. 1,
Block, C. S. 2 and Nir-Paz, R. 2
1. Meir medical center, Kfar-Saba, Israel
2. Hadassah Hebrew University Medical Center, Israel
3. Israel Ministry of Health, Central Laboratories
4. Tel-Aviv Souraski medical Center, Tel Aviv, Israel
5. Rambam medical center, Haifa, Israel
Listeria monocytogenes (LM) is a ubiquitous foodborne pathogen. Invasive infections
are rare, mainly affecting elderly people, the immunocompromised and
neonates. In pregnancy it causes fetal loss, preterm delivery and neonatal morbidity.
Although the incidence of listeriosis is low compared with other enteropathogens,
it carries a high mortality. Our aim was to characterize LM morbidity,
risk factors and molecular epidemiology in Israel to improve understanding
of the disease burden in Israel and assist public health policy-making. We performed
a nationwide retrospective study of all LM cases during 1998 – 2007. Cases
were actively sought in the records of all hospital-based clinical microbiology laboratories,
the national reference LM laboratory and LM cases reported to the district
physicians. 481 cases were identified, of which 166 were pregnancy-associated.
The yearly incidence peaked in 2006 and 2007 at 9.6 cases/million, which is
almost 5 times than the current rate in the USA. The incidence in pregnancy varied
between 5 and 25 cases/100,000 pregnancies/year. Annual rates above 15
cases/100,000 occurred several times. The perinatal case fatality rate was 47 %,
comprising a fetal loss of 38.2 % and additional 7.9 % neonatal mortality. Fetal survival
improved as pregnancy age advanced (P < 0.05). LM associated late abortion
occurred in 26.6 % and preterm labor in 46.8 %. A single maternal death was
recorded. The case fatality in non-pregnancy cases was 18 %. Additionally a gradual
increase in elderly occurred during the years and reached 42.7 cases/million/year.
Geospatial analysis revealed 2 districts with a higher incidence of listeriosis. Molecular
analysis (PFGE) of LM isolates suggested that a third of LM pregnancy associated
cases were caused by a single clone. In Israel, Listeria monocytogenes is an
important foodborne pathogen causing appreciable morbidity and mortality. Further
studies of the sources, molecular epidemiology and specific virulence determinants
should be undertaken.
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AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
ORAL PRESENTATIONS // O / 24–36
Human isolates of Listeria monocytogenes during
half a century in Sweden
Lopez-Valladares, G., Danielsson-Tham, M.-L., Vishal Singh, P. and Tham, W.
School of Hospitality, Culinary Arts & Meal Sciences, Örebro University, Grythyttan, Sweden
Over 800 isolates of Listeria monocytogenes have been collected from cases of invasive
listeriosis in Sweden – the first from 1958 and the latest from spring 2010.
All isolates are serotyped and characterized with pulsed-field gel electrophoresis
(PFGE) and Asc I restriction enzyme. From 1972 to 1995, serovar 4b was the predominant
serovar in human invasive listeriosis in Sweden. During the 1980s, some
listeriosis outbreaks caused by L. monocytogenes serovar 4b, belonging to a special
PFGE type, were diagnosed in the USA and Europe and were due to consumption
of dairy products. The predominant strain in listeriosis cases in Sweden during
the same time belonged to this type. The hypothesis is that a majority of human
listeriosis serovar 4b strains in Sweden came from soft cheeses imported from
Mediterranean countries. The hunt for L. monocytogenes serovar 4b in food production
plants is intensive in EU and USA and during recent years, cheeses have
had higher microbiological quality due to certified dairy farms and increased microbiological
control. This may be the reason for the decrease of serovar 4b cases
of listeriosis in Sweden. In 1996, serovar 1/2a became the major serovar in listeriosis
cases in Sweden. The consumption of gravad and cold-smoked salmons has
increased in Sweden, and in a recent study, 12.9 % of ready-to-eat vacuum-packed
gravad salmon and 28.0 % of cold-smoked salmons harboured L. monocytogenes.
The maximum number of L. monocytogenes was 1500 cfu/g product. The most
common PFGE types found in human cases of listeriosis in Sweden today are also
the types frequently encountered in vacuum-packed cold-smoked and gravad
salmon/rainbow trout.
Estimated incubation periods for listeriosis vary according to clinical
form of disease
Goulet, V., King, L., Vaillant, V. and De Valk, H.
Institut de Veille Sanitaire, France
Data on the incubation period of listeriosis are scarce. The incubation period can
be calculated precisely when a patient has had a single exposure to a confirmed
source of contamination. This information can be collected during the investigation
of point-source food borne outbreaks. Our study aimed to estimate the listeriosis
incubation period using available outbreak investigation data. Cases of invasive
listeriosis from confirmed food borne point-source listeriosis outbreaks
with precisely documented incubation periods and clinical forms of infection were
identified during the period 1985 – 2008 by literature review and from non-published
French outbreaks. Data from 9 published papers and from 6 French outbreaks
were analysed. A precise incubation period was documented for 28 cases: 13
with central nervous system (CNS) involvement (10 French outbreak and 3 published
cases), 15 pregnancy-associated cases (12 French outbreak and 3 published
cases). A confirmed food vehicle was identified for all outbreaks (rice salad, icecream,
cheese, meat-based products). The overall median incubation period was 17
days (range: 2 – 88 days). A longer incubation period was observed for pregnancyassociated
cases (median: 28 days (range: 14 – 88 days)) than for cases with CNS involvement
(median: 10 days (range: 2 – 19 days)). Insufficient data did not allow
for analysis of the incubation period of the bacteraemic form of infection. Our results
suggest that the incubation period for listeriosis varies according to the clinical
form of disease. A longer incubation period was observed for pregnancy-associated
cases than for cases with CNS involvement. This information could have
implications for the investigation of food borne listeriosis outbreaks as the incubation
period is used to determine the time period for which a food history is collected.
Adapting this time period according to the clinical form of infection could
facilitate a more precise identification of food products likely to be the source of
contamination.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
ORAL PRESENTATIONS // O / 24–36
Listeria monocytogenes in food and animals
in the European Union in 2008
da Silva Felício, M. T., Rizzi, V., Boelaert, F. and Makela, P.
Scientific Cooperation and Assistance Department, EFSA, Italy
The Directive 2003/99/EC on Zoonoses obligates the European Union (EU) Member
States (MSs) to collect data of zoonoses, zoonotic agents, antimicrobial resistance
and food-borne outbreaks. These data are reported to the European Food
Safety Authority (EFSA) who publishes the Community Summary Report. In 2008,
a large number of investigations (128,000) concerning ready-to-eat (RTE) foodstuffs
were reported by 26 MSs. The food categories most often covered were RTE
meat products, cheeses and fishery products. L. monocytogenes was seldom detected
above the legal safety limit of 100 cfu/g from RTE foods and findings over
this limit were most often reported from fishery products, cheeses, meat products
and sandwiches at levels of 0.2 – 0.5 % in the EU. L. monocytogenes was isolated
from both cheeses made from raw or low-heat-treated milk and pasteurised milk
as well as from soft/semi-soft and hard cheeses. L. monocytogenes was most often
detected in soft and semi-soft cheeses made from pasteurised milk. L. monocytogenes
was also reported, generally at a relatively low prevalence, from various animal
species in 2008, demonstrating that animals act as one reservoir of Listeria
bacteria although they rarely serve as a direct source of human infections. The
highest prevalence of L. monocytogenes was found in sheep, goats and cattle. Reported
data on the findings in RTE foods may be used to guide food controls carried
in MSs to ensure compliance with L. monocytogenes criteria. Compared to
previous years the quality of the data received improved as regards reporting of the
stage of sampling and the use of appropriate test methods that has eased the assessment
of compliance with Listeria criteria at Community level.
Listeria monocytogenes infection in the over 60s in England
between 2005 and 2008: a retrospective case-control study utilising
market research panel data
Gillespie, I. A., Mook, P., Little, C. L. and Grant, K.
Health Protection Agency, UK
Listeriosis is a rare but life-threatening foodborne disease and, therefore, the investigation
of factors which might alter people’s risk of infection is important to inform
on prevention and control. The incidence of listeriosis in England has doubled
since 2001, with a largely unexplained increase in people aged ≥ 60 years
presenting with bacteraemia without central nervous system involvement. Standardised
epidemiological data has been sought on cases of listeriosis reported in
England since 2005, but the value of the data accrued is limited without some
knowledge of exposure prevalence in the population at risk of listeriosis. The exposures
of listeriosis cases aged ≥ 60 years reported in England from 2005 – 2008
were compared to those of market research panel members representing the same
population and time period. Exposures were grouped to facilitate comparison.
Odds ratios and 95 % confidence intervals were calculated. Cases were more likely
than panel members to report cold cooked meats (beef and pork, but not chicken),
smoked fish (specifically salmon) and shellfish (prawns), dairy products (milk and
certain cheeses) and mixed salads. They were less likely to report the consumption
of sandwiches and fresh vegetables. To our knowledge this is the first time that
market research data has been applied to infectious disease epidemiology in this
way. The diversity of high-risk food exposures reflects the ubiquity of the microorganism
in the environment and/or the susceptibility of those at risk, and suggests
that a wider variety of foods can give rise to listeriosis. Food safety advice on
avoiding listeriosis should be adapted accordingly.
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AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
ORAL PRESENTATIONS // O / 24–36
Better and faster typing, MLVA – shall we play together?
Larsson, J. T. 1, Roussel, S. 2 and Moller Nielsen, E. 1
1. Statens Serum Institut, Copenhagen, Denmark
2. Agence Française de sécurité sanitaire des aliments, Maisons-Alfort, France
In 2007 and 2008 there were four papers published on the subject of MLVA (Multi
Locus VNTR [Variable Number of Tandem Repeats] Analysis) in L. monocytogenes.
During the same period a protocol with an extensive bioinformatic approach was
developed at SSI. The yet unpublished SSI MLVA protocol was created after analysis
of 20 genomes. Ten VNTRs were chosen on the basis of both nucleotide and
amino acid sequences analysis. The VNTRs have repeat lengths ranging from 6 to
15 basepairs. Degenerate primers were designed to match the diversity in the
genomes, taking good care not to include any gaps (which were present in several
loci/genomes). Finally three multiplex PCR groups were created and the VNTRs
were analysed by electrophoresis. The SSI method has been evaluated with the
last years Danish human listeriosis cases and the North American ILSI Listeria
collection. Results indicate a very close match to two-enzyme PFGE and an even
better separation of isolates. These results in concert with the reduced time and
cost for using MLVA for typing started off an incentive for unification and standardisation
of MLVA in L. monocytogenes. In a recently started cross laboratory
study, a comparison of the protocol published by Kate Sperry (2007) and the protocol
developed at SSI is under way. The methods are evaluated together with traditional
typing methods such as PFGE and agglutination serotyping. The MLVA
protocols are tested using capillary and agarose gel electrophoresis. Strain collections
includes; a selection of the WHO subtyping study from 1986 and a selection
from food and human isolates samples from France and Denmark respectively.
The chosen method might include a reduced set of loci and/or a mixture of the
two assays. The results from this cooperation will lead to a recommendation on a
MLVA protocol for use in Denmark and France.
comK prophage junction fragments in Listeria monocytogenes contain
SNPs that differentiate subclones of ECII and ECIII that are unique to
individual meat and poultry processing plants in the U.S.
Knabel, S. J.
Department of Food Science, Penn State University, USA
Many outbreaks of listeriosis in the U.S. are due to epidemic clones II and III, especially
those associated with ready-to-eat meat and poultry products. Both of
these epidemic clones have backbone genomes that are highly conserved, but contain
a hypervariable 40 Kb comK prophage. All isolates analyzed in the present
study that were suspected of being ECII or ECIII were first confirmed as such by
multiplex PCR and multi-virulence-locus sequence typing (Chen and Knabel,
2007) and included those from the 1998 – 1999 hot dog and 2002 turkey deli outbreaks
in the U.S., those from multiple states that were generated as part of USDA
FSIS's meat and poultry plant surveillance program, and those that were previously
isolated from two turkey processing plants in two different states (Eifert et
al. 2005). Upstream and downstream prophage junction fragments in all ECII and
ECIII isolates were amplified using 1/4 and 2/3 primer sets modified from Loessner
et al. (2000) and subsequently sequenced. The junction fragments in ECIII
isolates from a sporadic case in 1988 and from an outbreak in 2000 due to turkey
deli meat in the U.S. were also amplified and sequenced. Cluster analysis based on
junction fragment sequences revealed the presence of five subclones of ECII, the
ECIII subclone and the Canadian 2008 subclone, which were unique to individual
meat and poultry processing plants. However, the ECII subclone that caused the
2002 outbreak was found in three different processing plants, two of which were
originally associated with this outbreak. We speculate that evolution of these subclones
may be due to extensive recombination and subsequent niche-specific
adaptation acting on the comK prophage, which may represent a “Rapid Adaptation
Island” in Listeria monocytogenes. Genes that were unique to the comK
prophage were identified as putative “Adaptons” and their possible roles in colonization,
transmission between and within meat and poultry plants, and subsequent
contamination of foods and humans will be discussed.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
ORAL PRESENTATIONS // O / 24–36
Genetic diversity of Listeria monocytogenes measured by multiple-locus
variable-number tandem repeat analysis (MLVA)
Hyytia-Trees, E., Sabol, A., Graves, L. and Ribot, E.
Centers for Disease Control and Prevention, USA
Listeria monocytogenes is a genetically and phenotypically diverse organism, encompassing
at least three genetically distinct lineages that appear to differ in their
likelihood of causing human and animal disease. Classification of isolates into
these three lineages has been confirmed by different subtyping methods, and also
correlates with serotype classification. In the present study, 250 epidemiologically
unrelated isolates from 1981 to 2009 representing serotypes 4b (93), 1/2a
(70), 1/2b (57), 3a (4), 4c (4), 4a (2), 1/2c (2) and untypeable (18) were characterized
using multiple-locus variable-number tandem repeat analysis (MLVA). The
isolate set also included one isolate from each of ten well characterized outbreaks.
The data was analyzed in BioNumerics software and a dendrogram was constructed
by UPGMA clustering using the categorical coefficient. A minimal spanning
tree was also created using the Manhattan coefficient. In the minimum spanning
tree, a clonal complex was defined as a group of related patterns that differed
from each other at a single locus by one or two repeats. A total of 139 unique patterns
were detected among the 250 isolates. Thirty one MLVA patterns were associated
with more than one isolate, with the most common pattern including 16
isolates with isolation years varying from 1983 to 2007. With very few exceptions,
the isolates clustered according to their lineage. Nineteen clonal complexes were
detected, encompassing a total of 129 isolates. Two different serotypes (1/2a & 3a,
1/2a & 1/2c) shared the same clonal complex in two instances. Five of the ten outbreak
related isolates were located in three different clonal complexes that included
nine or more isolates with multiple isolation years and origins in different
continents. In conclusion, the MLVA scheme used in the present study is in accordance
with the currently used lineage classification and indicated the presence
of successful epidemic clones.
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AREA // D // Strategies for prevention and control of Listeria monocytogenes
ORAL PRESENTATIONS // O / 37– 46
Time Temperature Indicators can be used as an effective Risk
Management Tool for Listeria monocytogenes in Ready-To-Eat foods
Koutsoumanis, K., Vaikousi, H. and Costas, B.
Department of Food Science and Technology, School of Agriculture,
Aristotle University of Thessaloniki, Greece
The objective of the present work was to evaluate the effectiveness of Time Temperature
Indicators as risk management tools for Listeria monocytogenes in RTE
foods. In a previous study we presented a microbial Time Temperature Indicator
(TTI) system based on the growth and metabolic activity of a Lactobacillus sakei
strain. In the latter system, an irreversible color change of a chemical chromatic
indicator (from red to yellow) progressively occurs due to the pH decline as a result
of L. sakei growth and metabolism in a selected medium. In this study we show
that both the L. sakei growth and colour change of the microbial TTI system exhibit
similar kinetic responses with the growth of L. monocytogenes. With an appropriate
selection of the initial level of L. sakei in the system, the TTI end point
(time at which a distinct visual color change to the final yellow was observed) can
be adjusted in order to indicate a certain level of L. monocytogenes growth in the
food during distribution and storage. Thus, the use of the microbial TTI can assure
a maximum limit in the growth of the pathogen from production to consumption
time by informing the consumers when this limit is exceeded in a product
unit. The latter limit, which we call Growth Tolerance Criterion (GTC), can be
considered as a performance criterion for the growth of L. monocytogenes during
distribution and storage. The applicability of the microbial TTI in reducing consumer
exposure to L. monocytogenes from the consumption of pate was evaluated
in a simulation study. The TTI was appropriately adjusted to provide a GTC=3 logs
CFU/g. The concentration of the pathogen and the colour of the TTI at the time of
consumption were estimated with a probabilistic approach using Monte Carlo
simulation. The simulation results showed that the application of the TTI resulted
in a significant reduction of consumer exposure to L. monocytogenes. Assuming
that packages in which the TTI end point has been reached before consumption
are discarded, the predicted percentage of consumed products contaminated with
L. monocytogenes concentrations above 10 3 CFU/g was reduced from 5 % to 0.2 %
with the use of TTI.
Safety of salad leaves and herbs
Garland, C. D. 1 and Clark, A. 2
1. FWE Health, North Hobart, Tasmania, Australia
2. Houstons Farm, Cambridge, Tasmania, Australia
Comprehensive HACCP-based procedures have been implemented to prevent Listeria
monocytogenes contamination of RTE (ready-to-eat) salad leaves and herbs in
a commercial facility in Tasmania, Australia. Production has increased from 27
tonnes in 1995 to 2000 tonnes in 2009, with cultivation now undertaken at 3 field
sites and processing in a centralised factory certified to third party audited food
safety standards. The range of consumers is wide, including typical high-risk
groups. More than 1200 samples of RTE products have been tested since 1995 and
currently 300 RTE samples are tested at early and end of shelflife annually. To date,
L. monocytogenes has been detected in one RTE sample only, an outstanding result.
Control Points/Critical Control Points for minimising Listeria contamination in
field conditions are implemented for: lettuce transplants; cultivation soil; catchment
animals and wild life; irrigation water; harvesting; acceptance/rejection criteria
for raw ingredients; potable water; transport of harvest to factory. CPs/CCPs
in the factory relate to: restricted access; strict separation of work spaces into lowmedium-high
risk zones; temperature control; reduction of microbial loads; product
packing; staff training; sensory monitoring of product; physical and microbiological
monitoring. CPs/CCPs for transport to wholesaler and retailer include:
sealed box packing, visual checking; sealed vehicle; temperature control/data logger;
microbiological monitoring. Data will be presented to illustrate key microbiological
and physical CPs/CCPs relating to: animal access to the field, soil additives
and composts; surfaces, equipment, potable water, irrigation water, raw
ingredients and RTE products; factory temperature control (ambient, water, product);
removal of organisms by washing, centrifugation and infrared drying, and
disinfection by chlorination (with auto adjustment of free available chlorine and
pH); transport.

AREA // D // Strategies for prevention and control of Listeria monocytogenes
ORAL PRESENTATIONS // O / 37– 46
Environmental factors affect adhesion of Listeria monocytogenes
to inert surfaces through flagellum expression
Tresse, O.
INRA, France
Listeria monocytogenes could adhere to inert surfaces and form biofilms. Biofilms
of L. monocytogenes constitute eventually a reservoir for cross- or re-contaminations
of food products in food-processing plants. Biofilm initiation includes an irreversible
attachment of cells (adhesion) to inert surfaces. Adhesion is therefore
crucial for the biofilm development and subsequently for dissemination and persistence
of food pathogens. Adhesion assays from four to 101 L. monocytogenes
strains from diverse origins (food, food-processing environment and clinical cases)
were conducted in polystyrene and stainless steel 96-wells microtiter plates. Adhesion
was also assessed after cell cultivation at different pHs, NaCl concentrations
and temperatures. In parallel, flagella were observed after cultivation in the
different environmental conditions using optical microscopy. Three naturally aflagellated
strains, five environmental flaA mutant strains and one flagellum-paralyzed
strain were also tested to verify the contribution of flagella to adhesion. Results
indicated that attached cells were significantly higher at pHs greater or equal
to 6, at 8 °C and 20 °C or at 0 % and 6 % NaCl than at pH 5, at 37 °C or at 11 % NaCl.
Correlatively, flagella were not detected at pH 5, at 37 °C and at 11 % NaCl. This
result indicates that the temperature is not the only environmental factor that
could regulate flagellum expression in L. monocytogenes. NaCl concentrations and
pHs closed to non-growing conditions inhibit flagellum expression. Naturally aflagellated
strains and ∆flaA mutants adhered at the same level of strains cultivated
in flagellum inhibiting conditions. In addition, ∆flaA mutants adhered equally
while adhesion capability of the respective parental strains varied according to
the strain. Furthermore, adhesion was also affected in the flagellum-paralyzed
strain indicating that motility plays an important role in adhesion capability. In
conclusion, conditions of food conservation have an effect on adhesion capability
of L. monocytogenes strains through flagellum regulation and flagella are responsible
for adhesion variation among strains.
Shelf-life laboratory durability and challenge studies for
Listeria monocytogenes in ready-to-eat foods: a presentation of the
European technical guidance document intended for laboratories
Beaufort, A., Bergis, H., Cornue, M. and Lardeux, A.-L.
Agence Française de Sécurité Sanitaire des Aliments (AFSSA), France
Listeria monocytogenes is a foodborne pathogen that may grow at refrigeration
temperatures and may be present in ready-to-eat (RTE) foods. In annex I of regulation
(EC) No 2073/2005 on microbiological criteria for foodstuffs, a specific attention
to food safety criteria for L. monocytogenes in RTE foods is given and annex
II of this regulation specifies that food business operators (FBO’s) shall conduct, as
necessary, studies to evaluate the growth of L. monocytogenes that may be present
in the product during the shelf-life under reasonably foreseeable storage conditions.
But this annex does not describe the procedure to conduct such shelf-life
studies to ensure that the criterion of 100 L. monocytogenes/g is met over the entire
intended shelf-life of the product. The European technical guidance document,
intended for laboratories conducting shelf-life studies for L. monocytogenes in RTE
foods in collaboration with the FBO’s, provides recommendation on how to select,
how to implement and how to perform the test(s) required. It describes the microbiological
procedures for determining growth of L. monocytogenes using challenge
tests and durability studies. Challenge tests, defined as tests providing information
on the behaviour of L. monocytogenes artificially inoculated in the food,
can be performed with two different objectives: (i)Classify whether a food does or
does not support growth of L. monocytogenes; (ii) Quantify the growth of L. monocytogenes
by assessing either the growth potential (δ) or the maximum growth rate
(∝ max ). Durability studies allow an evaluation of the growth of L. monocytogenes in
a naturally contaminated food during its storage according to reasonably foreseeable
conditions. This technical document, prepared by the EU Community Reference
Laboratory for L. monocytogenes in collaboration with a working group consisting
of 10 laboratories, including nine National Reference Laboratories for L.
monocytogenes, is available since 15 December 2008 on the website of the European
Commission, DG Health and Consumers.
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AREA // D // Strategies for prevention and control of Listeria monocytogenes
ORAL PRESENTATIONS // O / 37– 46
Successful strategies against Listeria monocytogenes in Switzerland
Imhof, R.
Agroscope Liebefeld-Posieux Research Station ALP, Switzerland
In the aftermath of the massive Listeriosis outbreak in 1987 due to a soft cheese
made out of raw milk, the Swiss government decreed the creation of appropriate
means to prevent a repetition of such a case. Agroscope Liebefeld-Posieux Research
Station ALP was given the order to maintain a laboratory for the detection
of Listeria and to develop a nationwide Listeria monitoring programme (LMP) in
cooperation with the Swiss dairy industry. LMP covers about 70 to 80 % of the
cheese production of Switzerland. The aims are to detect contamination in cheese
production sites and ripening centres as early as possible, to stop the spreading
of Listeria by means of appropriate measures and above all to prevent contaminated
products to be placed on the market. The programme is legally based on private
law and guaranties confidentiality to its members. Although the results are
not open to the public, ALP closely works together with the concerned public authorities
in cases of enterprises with problems. ALP also created a special task
force: By incidence of problems with Listeria the ALP consulting team can be demanded
from any enterprise for analysis and advice as well as for direct participation
in the process of complete redevelopments. Following defined proceedings
the ALP team helps in cooperation with the enterprises own emergency team to
find individually designed solutions. Periodical controls in the following years
proof the success and the sustainability of interventions and worked out security
concepts. The history of outbreaks and the European RASFF notifications of the
last 15 years concerning Switzerland manifest the success of this specific approach.
Despite many ingenious efforts and product developments, there exists – to our
knowledge after 20 years of specific experience – no single measure or method to
solve a problem within the dairy food chain caused by L. monocytogenes. But observance
of the principles of Good Manufacturing Practice, a specifically designed
concept of security – which has to be lived by staff and personnel – besides to periodical
controls guarantee that every production site is capable to produce and
distribute safe and healthy dairy products.
Absence of Listeria monocytogenes growth during raw milk
cheesemaking: a modelling approach
Jordan, K. 1, Schvartzman, S. 1,2, Butler, F. 2 and Tenenhaus-Aziza, F. 3
1. Teagasc, Moorepark Food Research Centre, Ferrmoy, Cork, Ireland
2. Biosystems Engineering, School of Agriculture, Food Science and Veterinary Medicine,
University College Dublin, Ireland
3. CNEIL, National Interprofessionnal Center for Dairy Economy, Paris, France
The presence of Listeria monocytogenes in certain foods and the risk that this poses
to public health and food quality is still a problem. Currently, the field of food microbiology
focuses on obtaining data on the behaviour of microorganisms in food,
but the responses obtained provide little insight into the relationship between
physiological processes and growth or survival. This link can be made through
mathematical models. In a simple form, a mathematical model is a simple mathematical
description of a process. The application of mathematical models to food
microbiology has been developed in recent years and now constitutes a new discipline
named as Predictive Microbiology. However, most predictive models are
based on laboratory experiments in microbiological media under static conditions.
As such models tend to be inaccurate, we have undertaken our experiments in a
food system under dynamic conditions. Cheese was made with raw and pasteurised
milk deliberately contaminated with L. monocytogenes. Listeria was monitored
through the manufacture and ripening period of the cheese. The results showed
that L. monocytogenes did not grow during manufacture of raw milk cheese, but
did grow during manufacture of pasteurised milk cheese. The data obtained for
growth, survival and inactivation was modelled. The application of models that
can explain the behaviour of Listeria in cheese and the further predictions that
can be obtained from these models are useful for the improvement of ongoing research
on biotraceability and for the better understanding of the general behaviour
of these microorganisms under dynamic conditions, such as in dairy products.

AREA // D // Strategies for prevention and control of Listeria monocytogenes
ORAL PRESENTATIONS // O / 37– 46
A predictive model to set high pressure processing criteria for
Listeria monocytogenes inactivation on dry-cured ham
Bover-Cid, S. 1, Belletti, N. 2, Garriga, M. 1 and Aymerich, T. 1
1. IRTA. Food Technology, Spain
2. Università di Bologna, Italy
The aim of the work was to validate and use a predictive mathematical model
for the inactivation of Listeria monocytogenes on dry cured ham by high hydrostatic
pressure (HHP) processing, as a function of the technological parameters:
intensity, length and fluid temperature. The model was previously developed
from the inactivation data obtained after processing sliced dry-cured ham,
spiked with L. monocytogenes, at different HHP conditions according to a central
composite design: at 347 – 852 MPa; for 138 to 945 seconds; at 7.6 to 24.4 °C. The
best fitting and most significant polynomial equation indicated that pressure
and time were the most important factors determining the inactivation extent.
By contrast, temperature showed no significant influence within the assayed
range. The statistically significant quadratic terms of pressure and time indicate
the little effect observed below 450 MPa. Similarly, holding time longer than
10 min did not result in a meaningful reduction of L. monocytogenes counts. The
mathematical model was validated with data obtained from further experiments
within the assayed experimental domain, including data from international scientific
literature. The accuracy and bias factor were within the proposed acceptable
values demonstrating the suitability of the model for predictive purposes.
As a practical application of the validated model, the mathematical
equation was used to predict the HHP process conditions needed to ensure the
safety performance criteria established by different authors and organisations, in
terms of logarithmic reductions of L. monocytogenes loads. The general performance
criteria of 6 Log reduction, as equivalent to pasteurization, suggested
by the Food and Drug Administration would be achieved with treatments above
600 MPa, for instance applying 807 MPa for 5 min or 746 MPa for 10 min. More
product-specific performance criteria proposed, i.e. 4D and 2.39D, would be
achieved with 5 min treatments at 703 MPa and 613 MPa, respectively.
Application of a validated predictive model to prevent growth
of Listeria monocytogenes in ready-to-eat foods – importance
for product development and risk management
Mejlholm, O. and Dalgaard, P.
Seafood & Predictive Microbiology, Technical University of Denmark
Predictive microbiology is a most active research area within food microbiology
and many successfully validated models are today available and useful for assessment
and management of microbial food safety and quality. Recently, an extensive
growth and growth boundary model for Listeria monocytogenes was developed
and successfully validated for ready-to-eat meat, seafood, poultry and non-fermented
dairy products. This model is flexible and includes the effect of 12 environmental
parameters (temperature, NaCl/a w , pH, smoke intensity, nitrite, CO 2 ,
acetic acid, benzoic acid, citric acid, diacetate, lactic acid and sorbic acid) as well as
their interactive effects. It can predict the growth boundary and thereby combinations
of product characteristics and storage conditions that prevent growth of L.
monocytogenes and this is most important in relation to EU regulations and codex
guidelines. Nevertheless, growth boundary predictions alone are insufficient for
formulation of safe foods because the likely variability in product characteristics
and storage conditions must be taken into account. For this, stochastic models can
be applied together with distributions of the relevant environmental conditions.
Important parameters to control growth of L. monocytogenes in ready-to-eat foods,
however, are not yet included in those models. We here present a simple alternative
that allow variability in product characteristics and storage conditions to be
taken into account when conditions that prevent growth of L. monocytogenes are
predicted. This approach relies on validated cardinal parameter growth models.
The effect of interaction between environmental parameters is used to predict
both the growth boundary and interface conditions within the no-growth region.
Data from ready-to-eat foods will be used to show how these no-growth interfaces
can be predicted in a sufficient distance from the growth boundary to account for
variability in the environmental conditions.
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AREA // D // Strategies for prevention and control of Listeria monocytogenes
ORAL PRESENTATIONS // O / 37– 46
Application of predictive microbiology to control the growth
of Listeria monocytogenes – dairy products as an example
Lobacz, A.
Faculty of Food Sciences, University of Warmia and Mazury in Olsztyn, Poland
Predictive microbiology is a relatively new scientific discipline which is used for
modelling behaviour of microorganisms, mainly food borne pathogens, as a response
to environment. It is based on assumption that the reaction of bacteria on particular
environment factors is reproducible, and on the basis of researches and observations
made in past, it is possible to predict the microbiological behaviour in defined environment.
Many mathematical models, with regard to Listeria monocytogenes, have
been generated, which describe survival, inactivation or growth of this food borne
pathogen as a response to environmental parameters. Models are generated usually
in microbiological liquid media, although more often data are obtained in food products.
In the following paper usefulness of predictive models describing the growth of
L. monocytogenes is presented. Application of tertiary models – ComBase Predictor
(www.combase.cc) and Pathogen Modeling Program (www.ars.usda.gov) is also discussed.
Moreover, microbiological experiment was performed in order to evaluate
behaviour of L. monocytogenes in Camembert type cheese during ripening and storage
at following temperatures: 3, 6, 9, 12, 15 and 21 °C. Primary and secondary models
were generated and their performance was checked by mathematical and graphical
validation. Predictive microbiology is a new tool to microbiological risk assessment.
As a subdiscipline of food microbiology, it is an intensively developing field. Generated
and validated predictive models become more accurate and more often used in
food industry in order to eliminate food borne outbreaks.
Characterization of Food Alert for Listeria monocytogenes in France in
2008
Leclercq, A. 1, Dusch, V. 2, Salah, S. 2, Laurent, E. 3, Chenal-Francisque, V. 1, Thierry-Bled, F. 4,
Lecuit, M. 1, Goulet, V. 3 and Pihier, N. 2
1. Institut Pasteur, NRC and WHO-CC for Listeria, Paris, France
2. Direction Générale de l’Alimentation, Ministère de l’Alimentation, de l’Agriculture et de la Pêche, France
3. Institut de Veille Sanitaire (InVS), France
4. Direction Générale de la Concurrence, de la Consommation et de la Répression des Fraudes,
Ministère de l’Economie, de l’Industrie et de l’Emploi, France
Among measures established to assure a high level of protection of human health
and consumers, the European regulation EC 178/2002 indicates that food business
operators shall notify all foodstuffs not in compliance with the food safety criteria
for Listeria monocytogenes (Lm) described in regulation EC 2073/2005. A food alert
is established when a foodstuff presenting a risk is on the market, or when rapid action
(withdrawal or recall) is required. In the present study, our aim was to characterize
all these French food alerts (FAs) for Lm in 2008. In case of FA, competent
authorities asked the food business operators or/and inspection services to send Lm
food strains from own-checks and official control to the NRC for Listeria to obtain
their genoserotype and combined AscI/ApaI patterns by PFGE. Weekly, comparison
of microbiological characteristics of food and human strains were performed on
a period of 6 months and were sent to involved competent authorities and French
Institute of Public Health Surveillance (InVS) with the aim of possible complementary
investigation, if necessary. In 2008, 280 FAs for Lm have been registered, mainly
at the retail level (184), from own-checks (215) and official control (65). Among these
280 FAs, Lm enumerations on sample(s) of FAs have been performed in 260 FAs
[Range: 10-740,000 CFU/g]. Incriminated foods were mainly in decreasing order:
meat products (134 FAs), raw milk cheeses (47 FAs) and seafood/fish products (40
FAs). 179 FAs were only followed by food strains invoice [Range: 1-52 strains/FA; 100
FAs with a single strain associated], representing 673 food strains. Genoserotypes of
food strains were at 55 % IIa, 21 % IVb, 19 % IIb and 4 % IIc. Combined AscI/ApaI
patterns of strains varied from 1 to 27 per FA [137 showed single combined
AscI/ApaI pattern]. 60 % of combined AscI/ApaI patterns for FAs food strains have
been also observed for human strains in 2008. 84 FAs have food strains found microbiologically
similar to human strains but no FA was linked with human cases in
2008. Work is in progress for comparison of these data to 2007 and 2009 data. Even
if high food contamination were sometimes observed, FAs were not at the origin or
followed by human cases in 2008. In addition with the measures performed by the
food business operators in the context with their sanitary control plan, food management
measures linked to FAs may have contributed in the French situation of no
Lm outbreak since 2003.

AREA // E // Communication, risk perception and consumer practices – Social sciences in Listeria control
ORAL PRESENTATIONS // O / 47– 49
Efforts to update and perform health education on listeriosis
in Los Angeles County, California, by the County of Los Angeles
Department of Public Health
Guevara, R. E.*
County of Los Angeles Department of Public Health, USA
Listeriosis became a relatively well-known disease in Los Angeles County after the
large southern California listeriosis outbreak in 1985. However, 20 years later, the
tasks of keeping the general population and medical community properly educated
have not been easy with public health priorities shifting across topics such as bioterrorism
preparedness, MRSA, West Nile Virus, pandemic influenza, and most recently
H1N1 influenza. In 2005, health education efforts on listeriosis were specific to only
certain high-risk groups and needed a broader audience. Understanding the County
of Los Angeles Department of Public Health’s (DPH) health education mission to encompass
the general community, a multi-disciplinary team composed of epidemiologists,
public health nurses, and health educators produced new listeriosis education
materials. Public health messages were developed by those who conducted case investigations
by interviewing patients, relatives, and providers of listeriosis cases, and
by those who performed epidemiologic surveillance. A standard of attractiveness was
set before recruiting health educators to help design the new education materials.
Finding various images and pictures, health educators drafted different designs for
posters, flyers, and brochures. Public health nurses formed focus groups to help refine
health education messages, particularly for materials translated into Spanish. No
systematic measurement for success of the health education materials was established;
however, the materials continue to be used by DPH as various health care
providers and organizations request them, and even when H1N1 influenza became
the focus of public health in April 2009, listeriosis was the most common search term
in the DPH website. This presentation describes the approach the DPH used to more
effectively communicate the risks of listeriosis not just to those who are vulnerable to
the disease, but also to those who might know or even take care of them.
* Participation Supported by IUFoST
Awareness of listeriosis among Portuguese pregnant women
Mateus, T. 1,3, Maia, R. L. 2 and Teixeira, P. 1
1. CBQF / Escola Superior de Biotecnologia – Universidade Católica Portuguesa, Porto, Portugal
2. CECLICO / Centro de Estudos Culturais, da Linguagem e do Comportamento Faculdade de
Ciências Humanas e Sociais – Universidade Fernando Pessoa, Porto, Portugal
3. Escola Universitária Vasco da Gama, Coimbra, Portugal
The role of Listeria monocytogenes as a foodborne pathogen was definitively recognized
during the 1980s. This recognition was the consequence of a number of epidemic
human outbreaks due to the consumption of contaminated foods, in Canada, the US
and Europe. Listeriosis is especially severe in immunocompromised individuals such
as pregnant women. The disease has a low incidence, although this is undeniably increasing,
and a high fatality rate amongst those infected. In pregnant women listeriosis
may cause miscarriage, foetal death or neonatal morbidity in the form of septicaemia
and meningitis. Improved education concerning the disease, its transmission
and prevention measures for immunocompromised individuals and pregnant women
has been identified as a pressing need. The aim of this study was to evaluate the awareness,
in terms of food safety and listeriosis, of pregnant women in the last trimester of
pregnancy or with babies up to 3 months old. For this purpose 956 women were questioned
as to their knowledge and practices relating to what foods to avoid, good hygiene
and cooking practices. Also of interest were the sources of information on Listeria
spp. and listeriosis, and from whom and how women would like to receive such
information. Two hundred and twenty nine women said they had not avoided any kind
of food, whilst 306 said they had not made any particular changes to their food safety
related habits when they prepared and cooked meals. Four hundred and seventy seven
women considered that they had received enough information about nutrition during
pregnancy, and that this information had been acquired from their doctor, who was
also the person considered the most competent to give this information. A hundred
and seventeen women said they had heard about listeriosis, although only 58 of these
had been able to describe listeriosis symptoms. Women showed plenty of interest in
new information about listeriosis, and they would like doctors to provide it, as well as
by brochures and in the pregnancy advice book provided by the State Medical Service.
It appears that plan is required, to raise awareness amongst health professionals for
the need for food safety education for pregnant women.
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AREA // E // Communication, risk perception and consumer practices – Social sciences in Listeria control
ORAL PRESENTATIONS // O / 47– 49
The development and progress of a risk-based strategy
for the control of Listeria monocytogenes in New Zealand
Castle, M. and Crerar, S.
New Zealand Food Safety Authority
The New Zealand Food Safety Authority (NZFSA) has developed a risk-based strategy
for the control of Listeria monocytogenes. One of the objectives of this strategy
is ‘no increase in the reported incidence of foodborne listeriosis over the five year
period to 2013’. The strategy proposes a consistent and informed approach to managing
the risk from L. monocytogenes, through: (i) the categorisation of ready-toeat
foods in relation to their potential to support the growth of L. monocytogenes;
(ii) the commissioning of scientific studies and the development of tools to support
industry and NZFSA make risk based decision; (iii) the application of management
controls for industry using a risk-based approach; (iv) the implementation of
the principles in the Codex Guidelines on the Application of General Principles
of Food Hygiene to the Control of Listeria monocytogenes; (v) risk communication
for vulnerable consumers and food businesses. The strategy also recognises that
there is the potential for the increased incidence of listeriosis cases due to the impact
of an aging population and the greater availability and range of chilled readyto-eat
foods and the objective will therefore only be achieved by working with the
New Zealand food industry and consumers to enhance the control of L. monocytogenes.
The L. monocytogenes strategy was launched in 2008 and the initial response
to the risk management tools developed will be explored including uptake and response
from industry.

POSTER PRESENTATIONS

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AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Compartmentalization of IFN-gamma and IL-6 receptors signalling
in Listeria monocytogenes phagosomes: immune vesicles
Ramos-Vivas, J., Carrasco-Marin, E., Madrazo-Toca, F., Fernandez-Prieto, L.,
Rodriguez-Del Rio, E., Carranza-Cereceda, C. and Alvarez-Dominguez, C.
Hospital Santa Cruz de Liencres and FMV-IFIMAV, Spain
Cytokine activated macrophages kill Listeria monocytogenes within the phagosomes.
Here, we describe a novel listericidal Stat1 mediated signalling pathway involving
IFN-γ and IL-6. This pathway is compartmentalized into IFN-γ/IL-6
phago-receptosomes, innate immunity vesicles that link LM destruction with specific
immunity and IL-6 regulation. The Stat1-mediated signal triggered within
this compartment, links Rab5a with the activation of the lysosomal enzyme effector,
cathepsin-D, and with the induction of oxidative mechanisms such as the phox
regulators, p67phox/Rac2-GTP, and the iNOS. Three downstream events link these
listericidal IFN-γ/IL-6 phago-receptosomes with specific immunity: (i) a Stat1independent
transformation into MIIC-like vesicles regulated by Rab5a-GTP that
allows the acquisition of αβ SDS-stable MHC-II dimers, cathepsin-D, Lamp-2 and
Limp-2. (ii) A Stat1-dependent degradation of the Listeria immune-dominant antigen,
listeriolysin O by cathepsin-D. (iii) A downstream Stat1 signal to regulate IL-
6 production and drive a Th-1 cell-mediated immunity.

AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Antimicrobial susceptibility among Listeria monocytogenes isolates
from non human sources in France over a ten year period
Granier, S. A. 1, Moubarek, C. 2, Colaneri, C. 1, Roussel, S. 1, Courvalin, P. 2 and Brisabois, A. 1
1. AFSSA, France
2. Institut Pasteur, France
Listeriosis is public health concern since 1980’s. This food-borne infectious disease
has very high rates of hospitalization and fatality. It usually requires antimicrobial
treatment. Recommendations are: Penicillin G or ampicillin combined or not with
aminoglycosides. Antimicrobial resistance has increased among Gram positive bacteria
during the last past years but very few data are available for Listeria monocytogenes,
especially from non-human sources. In order to determine the frequency of
resistance within the species L. monocytogenes, a collection of 205 food and environmental
isolates, randomly selected among a collection of 10 year period
(1996 – 2006) was tested for antimicrobial susceptibility. This collection of non epidemiologically
related isolates included major serotypes (1/2a,1/2b,1/2c and 4b
strains) encountered, each of them presenting a unique PFGE profile. The phenotype
was determined using the microdilution method as recommended by CLSI.
Most of the strains presented a wild type phenotype: susceptible to penicillin G,
ampicillin, tetracycline, erythromycin, gentamicin, sulfonamides, trimethoprim,
vancomycin and resistant to 3rd generation cephalosporines, quinolones. Only four
strains displayed an original phenotype: two isolates (serotype 4b) were resistant to
erythromycin, two isolates (serotype 1/2a) were resistant to tetracycline, one of the
two last isolate was additionally resistant to trimethoprim. PCR analysis detected
the presence of erm(B) gene in the 2 erythromycin resistant strains, tet(M) in the
tetracycline resistant strains and dfr(D) was identified in the unique trimethoprim
resistant strain. Interestingly, int gene, involved in the mobility of conjugative
transposons, was found in the strains harboring tet(M) gene. The present results do
not indicate high frequency of resistance, nor large dissemination of resistance.
These data should now be compared to the one obtained for human isolates. Nevertheless,
those findings need to be regularly updated to make sure that the resistance
situation in L. monocytogenes doesn’t shift.
Five homologous small RNAs are involved in the response
of Listeria monocytogenes to cell wall acting antibiotics
Kiil Nielsen, P. and Kallipolitis, B.
Department of Biochemistry and Molecular Biology, Denmark
Small RNAs (sRNAs) are important regulatory elements involved in a variety of
cellular responses, including stress tolerance and virulence. In Listeria monocytogenes
five homologous sRNAs, named LhrC1-5, were identified recently by coimmunoprecipitation
with the RNA chaperone protein Hfq. The objective of the
present study is to improve our understanding of the role(s) of LhrC1-5 in L. monocytogenes.
Our study involves the identification of factors that influence the expression
of LhrC1-5 and investigations of genes affected by LhrC1-5. A range of
different environmental conditions and regulatory mutant strains were employed
to identify factors that affect the expression of LhrC1-5. The presence and stability
of the RNA transcripts were determined by Northern blotting. Furthermore, expression
of lhrC1-5 was followed by construction of promoter-lacZ fusions for all
five sRNA-encoding genes. These studies revealed a differentiated transcription of
LhrC1-5 in response to the presence of cell wall acting antimicrobial agents, such
as ethanol and β-lactam antibiotics. Cell wall acting agents have previously been
shown to activate the two-component systems LisRK and CesRK in L. monocytogenes.
Our experiments revealed that transcription of lhrC1-5 is completely dependent
on the presence of a functional LisRK system, whereas the CesRK system
appears to be important for a differentiated transcription of lhrC1-5 in
response to the above-mentioned cell wall acting agents. Finally, a mutant strain
lacking lhrC1-5 was constructed and characterized, and genes regulated by LhrC1-
5 were investigated by comparing the global gene expression of a wild type strain
vs. a ∆lhrC1-5 mutant strain. In summary, this study shows that L. monocytogenes
responds to cell wall acting agents through a complex gene regulatory network
which includes five small non-coding RNAs.
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AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Phenotypic analysis of selected listerial secretion mutants
Halbedel, S.*, Galander, S. and Flieger, A.
Robert Koch Institute, Germany
To be secreted into any extracellular compartment, proteins need to be translocated
across the cytoplasmic membrane first. Protein translocation is an energy
driven process and mediated by different sophisticated nano-machines that link
energy consumption to substrate translocation. Bulk protein translocation in the
Firmicutes is mediated by the Sec translocon together with the YidC proteins that
have their function in the lateral release of translocation substrates into the phospholipid
bilayer. Listeria monocytogenes YidC homologues are encoded by the spoI-
IIJ (lmo2854) and yqjG (lmo1379) genes. In addition, several minor secretion systems
have been described that transport certain subsets of translocation
substrates only, such as the Tat system, or that are supposed to be involved in protein
secretion since they seem to form proteinaceous membrane pores. Among
the latter is the Bacillus subtilis SpoIIIAH protein that recently was shown to be
the mother cell-specific part of a channel connecting the mother cell with the prespore
compartment in sporulating B. subtilis cells. A homologue of this gene can be
found in L. monocytogenes as well (lmo0791), whereas the prespore-specific component
(spoIIQ) and the other seven genes of the B. subtilis spoIIIA operon seem
to be absent from the listerial chromosome. L. monocytogenes SpoIIIAH consists of
an N-terminal transmembrane helix and a putative extracellular domain at the Cterminus
that shares similarity with the ring forming proteins of the YscJ/FliF
family associated with type III protein secretion. We therefore hypothesized that
SpoIIIAH might be involved in secretion of proteins or other biomolecules into
the extracellular space in L. monocytogenes. To test this hypothesis and to describe
the impact of YidC homologues on protein secretion we generated strains with
deletions of the spoIIIAH, yidC or yqjG genes. Here, we will present data describing
the phenotypic and proteomic analysis of these mutants.
* Participation Supported by IUFoST
Distribution of serotypes and pulsotypes of Listeria monocytogenes
in pig farms (France 2008)
Boscher, E.
AFSSA, France
This work was undertaken to estimate the prevalence of L. monocytogenes and, distribution
of serotypes and genotypes in French farrow-to-finish pig farms in 2008,
at the breeding pig level. A total of 730 faeces (10 per farm) were sampled from
sows in 73 pig farms localized in Brittany, France. Detection of L. monocytogenes
was carried out according to the ISO 11290-1/A1 method. Isolates were serotyped
and typed by PFGE. We found that 46.6 % of the farms (34/73) had at least one positive
sample and 10.7 % of the samples (78/730) were positive for L. monocytogenes.
A total of 125 strains were collected. Isolates belonged to four serotypes (1/2a, 1/2b,
4b and 1/2c) with a percentage of 41.6 %, 36.0 %, 20.8 % and 1.6 % respectively. Out of
the 34 positive farms, 19, 12 and 3 had respectively 1, 2, and 3 serotypes. Moreover,
21, 17, 11 and 1 positive farms had respectively the serotype 1/2a, 1/2b, 4b and 1/2c.
The genetic diversity of the isolates collected in this study was very important
(DI=0.986). In total, 50 genotypes were obtained; 25 for 1/2a, 15 for 1/2b, 9 for 4b
and 1 for 1/2c. Moreover 17, 6, 4, 3, 3, and 1 positive farms had respectively 1, 2, 3, 4,
5 and 6 genotypes. Furthermore, strains showing similar genotypes occurred in
several farms; as example, genotypes G18, G12 and G10 were found in 4, 5 and 5
farms respectively. This study provided recent valuable information on the occurrence
of L. monocytogenes in French pig farms. This microorganism is prevalent in
the sows in farrow-to-finish pig farms (46.6 %). Our work highlighted for some
farms several serotypes and genotypes suggesting several sources of contamination.
Furthermore, the serotypes found in this study are identical to those usually
involved in human listeriosis in Europe (Goulet et al. 2008).

AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Comparative phylogenomics of Listeria monocytogenes reveals
an adaptation profile
Silveira Nalério, E. 1, Padilha Silva, W. 1, Stabler, R. 2 and Wren, B. W. 2
1. Universidade Federal de Pelotas, Brazil
2. London School of Hygiene and Tropical Medicine, UK
Listeria monocytogenes is the causative agent of listeriosis which may cause a range
of diseases from gastroenteritis to death. In fact, disease outcome can be related to
strain serotype/lineage thus molecular analyses has demonstrated that L. monocytogenes
is a highly diverse species which can be grouped into three lineages.
Whole-genome microarray can be employed to study phylogenetic relationships
among Listeria strains either species or serotype level, in addition to demonstrate
differences on their virulence potential and/or environmental adaptation. The
aim of this study was the whole genome comparison of L. monocytogenes strains
from different origins. Ninety-nine L. monocytogenes strains from different geographical
origins (Brazil, Denmark, Austria, Ireland, USA and unknown), including
clinical strains (humans and animals), food and food industries strains were analyzed.
DNA from all strains were competitively hybridized on a L. monocytogenes
DNA microarray based on the whole-genome sequence L. monocytogenes EGD-e.
DNA labeling and hybridization protocol were followed according to Dorrell et al.
(2001). Data acquisition, processing and comparative phylogenomics were performed
as previously described by Stabler et al. (2006). Comparative phylogenomics
clustered the L. monocytogenes strains into two central clades which is
representative of the two main lineages of this species. In addition each of these
clades was divided into two further subclades. Clade formation was independent of
the geographical origin of strains with the exception of the clade containing persistent
strains (strains that persist in food-processing environment), where none
of the Brazilian strains were present. It was found 18 specific genes for lineage I
strains (1/2a and 1/2c serotypes). These genes are related to carbohydrate metabolism,
two component regulatory system, ABC transporter complex and bvrB and
bvrC genes. Significantly all persistent strains clustered together in the same lineage
I clade. We achieved a set of unique genes belonging exclusively to L. monocytogenes
persistent strains pointing to be responsible for its adaptation profile.
The genes are involved in stress resistance and are related to carbohydrate transport
and metabolism, environmental information processing, signal transduction
mechanisms, cell surface protein, amino acid transport and metabolism, nucleotide
transport and metabolism, translation, cell wall biogenesis, replication, recombination
and repair, transport of small molecules similar to ABC transporter,
metabolism of lipids and unknown function. Interestingly from 14 virulence listed
genes most of them were present in all studied L. monocytogenes strains with exception
of inlE and inlG genes. These findings indicate that genetic variability of L.
monocytogenes strains point to niche adaptation instead virulence differentiation
despite of different origins. Persistent strains clustered suggesting genetic origin to
survival in this environment.
Serotyping and PFGE patterns of Listeria monocytogenes isolated
from poultry meat
Vasantrao Kurkure, N. 1, Kalorey, D. R. 1, Rodrigues, J. 2, Gunjal, P. 1 and Barbuddhe, S. B. 2
1. Nagpur Veterinary College, Maharashtra Animal & Fishery Sciences University, India
2. ICAR Research Complex for Goa, India
Listeria monocytogenes is a gram positive, facultative, intracellular bacteria which is
ubiquitous in nature. Poultry meat and products frequently have been implicated in
outbreaks of food borne illness. In India there is practice to sell freshly slaughtered
poultry meat. A total of 119 poultry meat samples were collected from poultry meat
shops. These samples were processed for isolation of Listeria spp. by adopting standard
protocol. Out of 47 Listeria spp. recovered 17 isolates were confirmed as L.
monocytogenes by biochemical and in vitro pathogenicity test. The L. monocytogens
isolates were subjected to multiplex PCR based serotyping and PFGE analysis. All
the isolates were grouped as 4b, 4d and 4e serotypes. By PFGE analysis four Asc I
profiles were observed. Thirteen isolates were observed to be of similar profile indicating
homogenecity among the isolates recovered from poultry meat. Present
findings indicated the need of improvement in hygienic practices while slaughtering
and sell of poultry meat as L. monocytogenes is of zoonotic pathogen.
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AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Genotypic characterization of Listeria monocytogenes isolated
from fresh leafy vegetables
Warke, S. 1, Kalorey, D. R. 1, Umap, S. 1, Sonegaonkar, A. 1, Patil, V. 1, Kurkure, N V. 1
and Barbuddhe, S. B. 2
1. Nagpur Veterinary College, India
2. ICAR Research Complex for Goa, India
Listeria monocytogenes is a food borne pathogen responsible for listeriosis, an illness
characterized by meningitis, encephalitis, abortion and septicemia in human
beings. A total of 417 samples comprising eleven different types of fresh leafy vegetables
were collected from supermarkets and local markets in and around Nagpur,
Central India to detect L.monocytogenes. The vegetables included spinach (78),
Fenugreek (82), Green Sorrel (22), Coriander (53), radish (57), amaranthus (30),
sour greens (12), lettuce (33), Mint Leaves (43) and dill leaves (07). All samples
were processed for isolation of L. monocytogenes by two step enrichment followed
by plating on selective media. Confirmation of the isolates was on basis of biochemical
characters, haemolysis on blood agar and Christie, Atkins, Munch Petersen
test (CAMP). The isolates were also tested for the virulence associated
genes namely, hlyA, plcA, actA, iap multiplex and prfA by PCR. A total of 31 (7.43 %)
isolates of L. monocytogenes were recovered from spinach (11), fenugreek (9), coriander
(3), radish (4), amaranthus (2) and mint leaves (2). All the genes were detected
in seven strains, four genes (hlyA, actA iap and prfA) in three strains. The
hlyA, actA and iap in six strains. Three genes (hlyA, iap and prfA) were detected in
five strains. The hlyA and iap genes were detected in ten strains. Fourteen isolates
were subjected to multiplex PCR serotyping and all belong to 4b, 4d and 4e
serogroup. Contamination of vegetables with L.monocytogenes is a cause of concern
as most of the vegetables are consumed raw as salad. Thus, there is increased
risk for transmission of listeriosis. Therefore proper washing and cleaning of fresh
leafy vegetables is recommended at harvesting and consumption.
Comprehensive appraisal of the exoproteome of Listeria monocytogenes
by genomic and proteomic analyses
Desvaux, M., Dumas, E., Chafsey, I., Chambon, C. and Hébraud, M.
INRA, France
Secreted proteins are the main tools used by bacteria to interact with their environment
and are relevant to the bacterial lifestyle. By definition, secreted proteins
are proteins actively transported via secretion systems but they can have
radically different final destinations. In monoderm (Gram-positive) bacteria, such
secreted proteins can (i) anchor to the cytoplasmic membrane, (ii) associate with
the cell wall, or (iii) be released into the extracellular milieu and are the called exoproteins.
The subset of proteins localized in the extracellular milieu constitutes
the extracellular proteome, also called exoproteome. Listeria monocytogenes is a
bacterial species both pathogenic and saprophytic, which exhibits a highly variable
level of virulence from one strain to another and such biodiversity might express
within the exoproteome. The present investigation aimed at developping of
a generic genomic strategy to predict exoproteins in monoderm bacteria as well as
determining those differentially expressed within a panel of representative strains.
Genomic analysis was performed following a novel rational bioinformatic approach
to predict exoproteins, which expression was determined by proteomic
analyses following 2-DE and identification by MALDI-TOF MS. The rational bioinformatic
approach integrates the structural properties of the proteins potentially
secreted as well as the respective secretion systems involved. Comparison of theoretical
and experimental 2-DE maps provided a comprehensive picture of the exoproteome.
Exoproteins were further discriminated between those found in all or
only in a subset of L. monocytogenes strains. While the core exoproteome included
proteins related to virulence and cell wall biogenesis, variation in the protein
members of these categories constituted the variant exoproteome. The novel and
rational in silico strategy here developed to predict exoproteins is adaptable and
fully applicable to other monoderm bacteria. This proteomic analysis resulted in
the first definition of the core and variant exoproteomes of this species and provides
auxiliary insight in bacterial physiology.

AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Investigating differences in lineages of Listeria monocytogenes
using comparative genomics
McIlwham, S., Farber, J. and Pagotto, F.
Bureau of Microbial Hazards, Canada
Listeria monocytogenes causes foodborne listeriosis and is ubiquitous in the environment.
Strains are often classified into lineages I (food); II (human illness); and
III (food-production). It is not clearly understood how lineages develop a tropism
for a particular niche. Using comparative genomics, we attempted to identify
unique genetic attributes within lineages that contribute to the tropism of the
pathogen. A mixed genome array, created from a genomic library of 15 L. monocytogenes
isolates, was used to compare the genomes of different strains. An isogenic
mutant for a glycoside hydrolase family 65 (GH65) was created based on microarray
screening. Forty-five strains from different sources were studied in Mueller-
Hinton broth with ampicillin, vancomycin or acriflavine. Additionally, the growth
rates of eleven strains in Listeria Enrichment Broth (LEB) were compared. A
GH65 gene was found to be present in lineage II isolates but absent in lineage I isolates.
A GH65 isogenic deletion mutant and other lineage I isolates showed a reduced
ability to proliferate in the presence of vancomycin and ampicillin. When
growth rates were compared in LEB, the GH65 deletion mutant behaved similarly
to lineage I strains. The GH65 gene, present in lineage II strains, may provide a
competitive advantage in the host environment. The difference in growth rate of
the lineage I and II strains in LEB may provide insight as to why certain serotypes
are more readily isolated from food commodities.
Autolysis in Listeria monocytogenes – a proteomic approach
Pinto, E. 1, Marques, N. 2, Andrew, P. W. 3 and Faleiro, M. L. 1
1. Instituto de Biotecnologia e Bioengenharia, Centro de Biomedecina Molecular e Estrutural,
Universidade do Algarve, Portugal
2. BioFig, Universidade do Algarve, Portugal
3. Department of Infection, Immunity and Inflammation, University of Leicester, UK
Autolysins play a role in many cellular processes including cell growth and division,
cell wall turnover, motility and also pathogenicity. Several growth conditions
may interfere with cell wall synthesis, leading to unrestricted activity of the
autolysins which can degraded the cell wall to levels that compromise the cell integrity.
During our work differences between Listeria monocytogenes strains in
the tendency to undergo autolysis was found. The objective of the present study
was to test the hypothesis that as cells enter stationary phase the pattern of protein
expression in autolytic and non-autolytic strains is different. To achieve this,
two L. monocytogenes strains (an autolytic [C897] and a non-autolytic [EGD])
were grown in a defined medium at favourable (30 °C) and non-favourable (20 °C)
autolysis growth conditions. The bacterial cells and the supernatant samples were
collected immediately before autolysis. The samples were subjected to a proteome
analysis. Remarkably at favourable autolysis condition the autolytic strain expressed
a series of stress proteins and was depleted of 2-C-methyl-D-erythritol 4phosphate
cytidylyltransferase an essential component of the 2-C-methyl-D -
erythritol 4-phosphate (MEP) pathway. Injuries in this pathway may cause a
negative impact on cell wall synthesis. In both proteomes of the non-autolytic
strain EGD and the autolytic strain C897 in non favourable autolyis conditions revealed
a higher expression of proteins involved in the energy generation, amino
acid and fatty acid metabolism. In the secretome of C897 the p60 protein was
over expressed at 30 °C but no significant differences were found between the
strain C897 and EGD. However p60 levels were higher in the autolytic strain intracellular
proteome when grown at 20 °C. The levels of the autolysin p45 were
higher in the secretome of C897 at 30 °C and significant differences between the
two strains were found.
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AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Role of flhA, cheR and motA in growth of Listeria monocytogenes
at low temperature
Mattila, M., Lindström, M., Somervuo, P. and Korkeala, H.
University of Helsinki, Finland
Temperature-dependent induction of flagella is a well-characterized phenomenon
in L. monocytogenes. At 37 °C L. monocytogenes is virtually non-motile and the motility
genes are down-regulated whereas at 30 °C and below the strains are highly flagellated
and motile. However, the essentiality of increased flagellum production during
growth at low temperatures is still unclear. To study this relationship, we
prepared knock-out mutants of three motility and chemotaxis genes, flhA, cheR, and
motA on L. monocytogenes strain EGD-e. We compared the expression levels of these
genes in cultures grown at 3, 25 and 37 °C by using qRT-PCR. The role of these genes
in the cold adaptation of L. monocytogenes was also studied with growth curve analysis,
motility assays and electron microscopy. The relative expression of flhA, cheR
and motA in EGD-e cells grown at 3 °C was significantly (p < 0.01) increased compared
to 37 °C. At 3 °C the level of flhA and cheR transcripts was also significantly
(p < 0.05) higher than at 25 °C. The growth curve analysis showed that in cultures
grown at 3 °C both the growth rates and maximum optical densities at 600 nm were
significantly (p < 0.001) lower for the mutant strains ∆flha, ∆cheR and ∆motA compared
to the wild-type strain. At 37 and 25 °C we did not detect any significant differences
between the wild-type strain and the mutants. The motility assays showed
that ∆flhA and ∆motA strains were completely non-motile at all three temperatures
whereas ∆cheR showed similar motility pattern to the wild-type at both 3 and 25 °C.
At 37 °C the motility of ∆cheR mutant and the wild-type strain was negligible. The
results suggest that flhA, cheR, and motA have a role in the cold tolerance of L. monocytogenes
strain EGD-e, and that flagellum production and chemotaxis are linked to
cold adaptation of L. monocytogenes.
The effect of acetic acid (at pH 5.5) or benzoic acid (at neutral pH)
on lipid composition and fluidity of Listeria monocytogenes membrane
Ioannis, D. 1, Anita, B. 1, Eleni, S. 2 and Mastronicolis, S. 1
1. Food Chemistry Laboratory of Chemistry Dept, University of Athens, Panepistimiopolis Zografou,
Athens, Greece
2. National Hellenic Research Foundation, Institute of Organic and Pharmaceutical Chemistry, Athens,
Greece
Listeria monocytogenes is able to alter its membrane fatty acids (FA) composition or
the composition of the polar group of the lipid molecules after a reduction in pH
(due to Acid Tolerance Response, ATR). The lipid changes might play a protective
role upon (acidic) stress decreasing the membrane fluidity and thus obstructing the
entrance of the stressful compounds. The aim of our work was to investigate the
modification of L. monocytogenes membrane lipids molecules (neutral, NL or polar,
PL lipids) and also their FA composition in response to low (acetic acid) or neutral
pH (benzoic acid) and their consequences on membrane fluidity. L. monocytogenes
(DP-L1044, D. Portnoy, University of Pennsylvania) total lipids,TL extracted from
stationary phase cells cultured (1L BHI broth, 30 °C) with low initial pH (5.5) by
acetic acid, Lm AA (optical density OD 600 =0.210±0.031, 72h, n=12) or with neutral
pH by benzoic acid (1g/L), Lm BA (OD 600 =0.682±0.013, 10h, n=9) and compared with
those of optimal conditions cells, Lm control (OD 600 =0.816±0.032, 10h, n=8). The results
revealed: i) Only acetic acid enhances the antimicrobial activity. ii) The sums
of straight saturated chain FA(16:0 and 18:0) and unsaturated FA(16:1 and 18:1)
were similarly increased, at the expense of branched chain FA(a-15:0 and a-17:0) of
each TL acidic adapted culture (increase of high-melting FA). iii) Singularly acetic
acid adaptation in L. monocytogenes was correlated with a higher content (49.3 %
among TL) of neutral lipids class than those of Lm control . Moreover, we correlated
the lipid changes with thermodynamic analysis (Differential Scanning Calorimetry,
DSC) of lipids in order to estimate experimentally the initial hypothesis, that both
adaptation mechanisms (the high-melting point FA increase and the NL accumulation)
would promote the decrease of membrane fluidity. The results revealed that
in each culture Lm BA and Lm AA TL was increased the phase transition temperature,T
c (5.3 °C and 3 °C) and enthalpy difference, ∆Η (10.2 % and 53.8 % respectively)
than Lm control . The thermodynamic findings confirmed the above hypothesis
of decreasing fluidity. The high ∆Η value of Lm AA (high energy amount to
change its lipids) is interpreted by NL accumulation.

AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Elucidation of the responses to weak acids in the human pathogen
Listeria monocytogenes using gene microarrays
O’Byrne, C. 1, Heavin, S. 1 and Morrissey, J. 2
1. NUI Galway, Ireland
2. University College Cork, Ireland
Contamination of processed food with Listeria monocytogenes is a major human
health risk. Food preservation regimes often include the use of weak organic acids
to reduce microbial growth. Although the response of L. monocytogenes to strong
acids has been investigated extensively, there little is known about how L. monocytogenes
responds to weak acids, particularly at the molecular level. The objective
of this study was to investigate the molecular response of L. monocytogenes to five
food-grade organic acids, acetic, lactic, sorbic, citric and benzoic acid. Careful
growth experiments were carried out to determine the inhibitory concentrations
of each of these acids on the growth of L. monocytogenes. Microarray and quantitative
RT-PCR experiments were then performed to elucidate the molecular responses
of the organism to weak acids. In total 222 genes were found to be differentially
expressed in response to at least one of the weak acids. The temporal
response of 9 of these genes to sudden exposure to lactic acid or citric acid was
studied using RT-PCR. These data highlighted the distinct responses observed to
the 2 weak acids and identified genes that showed strong unique transcriptional
responses (both up-regulation and down-regulation) to each. At a global level there
was surprisingly little overlap between genes that responded to each acid. For example,
the 7 genes differentially expressed in the presence of acetic acid were not
influenced by any of the other acids. Together the data suggest that each organic
acid influences the cell in a very specific way, a finding that suggests that each acid
has a different inhibitory mode of action.
The immunogenic surface protein IspC acts as an
N-Acetylglucosaminidase in Listeria monocytogenes serotype 4b
Ronholm, J.*
University of Ottawa, Canada
IspC is a surface protein isolated from Listeria monocytogenes serotype 4b which
acts as an autolysin and virulence factor. Several autolysins including P60, Ami
and Auto have been isolated from various L. monocytogenes serotypes but the role
for their apparent functional overlap remains unknown. Further characterizing
these autolysins may help to gain insights into their roles in Listeria biology. The
novel autolysin IspC remains understudied. A panel of 15 monoclonal antibodies
(mAbs) directed against the surface of L. monocytogenes serotype 4b were generated,
characterized as recognizing IspC and will facilitate further study of this protein.
Based on serological assays with these mAbs we have determined that IspC
expression is limited to certain L. monocytogenes serotypes including 4a, 4ab, 4b,
and 4e, although, other serotypes have genes highly homologous to IspC. Sequence
homology to other autolysins predicted IspC functioned as an N-acetylmuramoyl-
L-alanine amidase. However, mass spectrometry analysis of peptidoglycan (PG)
hydrolysis with IspC has shown that IspC acts as an N-acetylglucosaminidase.
Based on the site of PG cleavage, it was hypothesized that the amount of PG acetylation
would affect IspC mediated PG hydrolysis. PgdA, the only one of three PG
deacetylases predicted to localize to the cell surface, is required for de-acetylation
of PG, which is assembled fully acetylated. To determine how acetylation affects
IspC PG hydrolysis, a pgdA deletion mutant was created. The mutant PG
(fully acetylated) was much more susceptible to hydrolysis by IspC than PG from
the wild-type, as assessed by re-naturing SDS-PAGE. Currently we are making a
pgdA complement to restore PG deacetylation in the deletion mutant. Further investigation
is being done on IspC hydrolysis of PG from the deletion mutant using
mass spectrometry. This will provide insight into the effects of the pgdA deletion
on PG structure and IspC PG hydrolase activity.
* Participation Supported by IUFoST.
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AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Listeria monocytogenes EGD chitinolytic activity is regulated by
carbohydrates but also by the virulence regulatory gene, PrfA
Halberg Larsen, M., Leisner, J. J. and Ingmer, H.
Faculty of Life Sciences, University of Copenhagen, Denmark
Chitin, a highly insoluble polymer of N-acetyl-D-glucosamine (GlcNAc), is a major
component of fungal cell walls and insect and crustacean exoskeletons and is one of
the most abundant polymers in marine and terrestrial environments. Chitin hydrolysis
by Listeria monocytogenes depends on two chitinase encoding genes chiA
(lmo1883) and chiB (lmo105) and the aim of this study was to investigate their regulation.
The production of the chitinases is substrate regulated and subjected to
catabolite control. Thus, chitin induces expression of both chitinases however chiA
but not chiB is furthermore induced by the monomer GlcNAc. In growth medium
supplemented with chitin and glucose the expression of both chitinases is repressed.
Expression of chiA and chiB is growth phase dependent with higher expression in
late exponential growth phase compared to early exponential phase. Chitinases expressed
by bacterial pathogens have proven to be important not only for nutrient
acquisition and environmental survival but also for infecting humans and animals.
Interestingly, we found that the central L. monocytogenes virulence gene regulator,
PrfA, is required for the chitinolytic phenotype as chitinase activity was significantly
reduced in ∆prfA mutant cells compared to the wild type cells. In agreement with
this result northern blot analysis showed that the amounts of chiA and chiB transcripts
were significantly lower upon induction by chitin in the ∆prfA mutant compared
with the wild type. Furthermore, in contrast to the wild type, no chiA transcript
could be detected in the mutant lacking PrfA during growth in medium
supplemented with GlcNAc. Regulation of chitinolytic activity in L. monocytogenes
is complex and the results obtained in this and other studies indicate that the biological
role of this activity may not be limited to the external environment.
Infectious dose curves for guinea pigs challenged with a Listeria
monocytogenes epidemic clone strain and a strain carrying a naturallyoccurring
virulence-attenuating mutation in inlA show a significant
shift in median infectious dose
Nightingale, K. 1, Van Stelten, A. 1, Simpson, J. M. 1, Chen, Y. 2, Scott, V. N. 3,
Ross, W. H. 4, Whiting, R. C. 5 and Wiedmann, M. 6
1. Colorado State University, USA
2. Grocery Manufacturers Association, USA
3. U.S. Food and Drug Administration, USA
4. Health Canada
5. Exponent, USA
6. Cornell University, USA
Listeria monocytogenes contains two subpopulations, including (i) epidemic clone
(EC) strains, which have been linked to the majority of listeriosis outbreaks worldwide
and are overrepresented among sporadic cases in some countries along with
(ii) strains carrying mutations leading to a premature stop codon (PMSC) in inlA,
which are commonly isolated from ready-to-eat foods (approx. 45 %) but rarely associated
with human disease (approx. 5 %). The virulence factor Internalin-A (InlA;
encoded by inlA) binds certain isoforms of the cellular receptor E-cadherin to facilitate
crossing of the intestinal barrier by L. monocytogenes. The current
FDA/FSIS/CDC L. monocytogenes risk assessment was developed with dose response
data from murine challenge experiments, a model that fails to probe InlA
mediated virulence due to the inability of InlA to bind the murine isoform of E-cadherin.
Guinea pigs, which express the human isoform of E-cadherin that binds InlA,
were intragastrically challenged with (i) a fully-invasive EC strain associated with a
listeriosis outbreak or (ii) a strain carrying the most common inlA PMSC mutation.
Dose-response curves for tissue infectivity were constructed with either a log-logistic,
beta poisson, or exponential (for spleen data) fit to the raw individual and combined
organ data. The log logistic and beta poisson models based on combined organ
data showed an approx. 1.3 log10 CFU increase in the median infectious dose for the
strain carrying a PMSC in inlA relative to the EC strain. Inclusion of strain effect
significantly improved the ability of the model to explain the observed data, supporting
a significant difference in tissue infectivity between EC and PMSC strains. L.
monocytogenes strains thus show notable differences in infectious dose required to
establish an infection. Results from this work support the dose-response relationship
for L. monocytogenes is strain-specific and will provide critical data for enhancement
of existing and development of future risk assessments.

AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
MudPIT based proteomic analysis of alkaline adapted, environmentally
persistent Listeria monocytogenes strains
Nilsson, R. E., Ross, T. and Bowman, J. P.
University of Tasmania, Australia
Soluble protein expression profiles of environmentally persistent and sporadic
Listeria monocytogenes strains in mid-exponential and stationary growth states
at pH 7.3 and adapted to pH 8.5 were compared using multidimensional protein
identification technology (MudPIT). Searches employed the X!Tandem algorithm
and an in house data validation criteria was applied using the Trans Proteomic
Pipeline (Version 3.4) and comparison against a decoy database. Qualitative and
relative abundances were compared through functional ontology (TIGR) and determination
of spectral abundances. A total of 1661 proteins were identified following
integration analysis. Significant differences in protein expression were observed
for both growth state and culture condition. Increased presence and
abundance of proteins associated with the cell wall, transport and binding, amino
acid / cofactor biosynthesis and protein stabilization were identified. The study
findings suggest alkaline adapted L. monocytogenes strains may use cytoplasmic
acidification by surrogate proton sources (both endogenous and exogenous) as a
means of persisting in high pH environments. Furthermore, proteins associated
with stabilization of cellular processes and cell wall modification may be aiding
in minimizing cellular damage under these conditions. This trend was identified in
both growth states; however it was more pronounced in exponential phase. No
significant difference was observed between the L. monocytogenes strains studied.
The results of this work support the notion that exposure to conditions favouring
alkaline homeostasis may be an important contributor to the development of persistent
L. monocytogenes, independent of strain.
RpoN, the alternative sigma factor, is associated with the growth phase
transition and pathogenesis in Listeria monocytogenes
Okada, Y., Suzuki, H., Monden, S., Igimi, S. and Okada, N.
Ministry of Health, Labour and Welfare, Japan
Listeria monocytogenes has 5 types of sigma factor. RpoN, an alternative sigma factor,
is involved in the bacteriocin-resistance and the osmotolerance in this bacterium,
however, its’ function remains almost unknown. We found that the rpoN
deletion mutant showed higher growth than its parental strain at the stationary
phase. To identify the RpoN regulated genes which are associated with the growth
phase transition, DNA microarray analysis was performed. From L. monocytogenes
strain EGD and its rpoN in-frame deletion mutant, total RNA samples were isolated
at early exponential and stationary phase. The gene expression profiles in
both strains at each growth phase were compared, and in the results, especially, 27
genes associated with oxidoreductase showed the different patterns of expression
between two strains. So we examined the survival of these strains after exposure to
the sub-lethal concentration of hydrogen peroxide. Both strains showed the similar
levels of tolerance against 100 mM hydrogen peroxide at stationary phase. However,
the mutant was significantly tolerant to hydrogen peroxide than EGD at the
mid exponential phase. From the result of electrophoresis, the chromosomal DNA
was degraded after exposure to hydrogen peroxide only in EGD, but not in the mutant.
Next, we examined the pathogenicity of the rpoN mutant. The mutant
showed the higher growth in J774 cells and the peritoneal adherent cells from
BALB/c mice. In spleen and liver of mouse, the mutant was able to grow at the
similar level of EGD, however, the growth was slowly. These results suggest that the
rpoN mutant is able to grow higher than the parental strain at the stationary phase
because of the increased DNA stability against the active oxygen accumulated in
the bacterial cells as a result of the respiration during the exponential growth, and
RpoN is associated with the pathogenicity of L. monocytogenes.
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AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Cellular lipid fatty acid pattern differences between reference and
ice-cream isolate of Listeria monocytogenes as response to cold stress
Anita, B., Ioannis, D. and Mastronicolis, S.
Food Chemistry Laboratory, Department of Chemistry, National University of Athens, Greece
One of the salient features of L. monocytogenes as a food-borne pathogen is its
ability to grow at low temperatures, allowing refrigeration at 5 °C to act as an effective
enrichment for the organism. There is little information about its ability to
withstand in frozen foods. L.monocytogenes is characterized by > 85 % branchedchain
fatty acids (FA), anteiso-15:0 (a-15:0) and anteiso-17:0 (a-17:0). Cells grown in
the cold (5 °C) contain significantly less a-17:0 than those grown at higher temperatures.
The purpose was to investigate the effect of ice-cream storage in
L.monocytogenes FA profile, after inoculation of pilot ice-cream with reference
strain DP-L1040 (D. Portnoy, University of Pennsylvania). The inoculation was
performed with reference strain Lm ref , (BHI, 30 °C, 8h, OD 600 =0.8) in order to
yield 6.30 log cfu/g ice-cream. Cells of L. monocytogenes isolated from inoculated
ice-cream, (Lm ice ), after 6 month of storage and cells of Lm ref were grown in BHI
broth, until late exponential phase at two different temperatures, at 30 °C (10h,
OD 600 =0.8) and at 5°C (8d, OD 600 =0.6). Each culture (Lm ice30°C , Lm ice5°C ,
Lm ref30°C and Lm ref5°C ) total lipids were extracted and FA methyl esters were analyzed.
Our results show that significant differences exist between the FA profile of
Lm ref30°C and Lm ice30°C . The Lm ice30°C was characterized by a-15:0 and a-17:0 FA.
Additionally, increase of straight chain 18:0 FA, (from 0.7 to 3.9 %) was observed in
cells of ice-cream isolate. There was also an increase of unsaturated FA. The
Lm ice5°C showed an a-15:0/a-17:0 ratio of 11.2, while Lm ref5°C was characterized
by significantly higher ratio of 19.3. Furthermore, there was a decrease (3.2 fold) of
∑branched/∑straight chain FA ratio in ice-cream isolate compared to reference
strain. These results showed that L. monocytogenes strain origin needs to be considered
when physiological studies are performed with this bacterium. The food
matrix and environmental conditions can influence the FA pattern and L. monocytogenes
survival. A greater understanding of cold adaptation mechanism may
offer methods for controlling L. monocytogenes in chilled and frozen foods.
Role of the dihydroxyacetone metabolism in the resistance of Listeria
innocua to pediocin
Milohanic, E.
INRA, France
Listeria monocytogenes is a Gram-positive bacterium, the causative agent of listeriosis,
which affects humans and animals. This bacterium which is a recurring contaminant
of the food chain represents a major problem for the food industry. We
were interested in the dha system of Listeria innocua that involves the general PTS
enzymes, the main transport and phosphorylation system of carbohydrates, which
also occurs in L. monocytogenes. It catalyses the intracellular PEP-dependent phosphorylation
of dihydroxyacetone (Dha). In silico studies allowed us to hypothesize
that the last gene of the dha system, pedB, could encode resistance to pediocin, a
bacteriocin produced by the lactic acid bacterium Pediococcus acidilactici. We
sought to understand why this gene is associated with the dha system. We first confirmed
the operon organization of this system. Quantitative PCR experiments revealed
that the expression of the dha system seems to be partially constitutive. Elevated
dha expression was observed in a dhaR mutant, which lacks the repressor of
this operon. We also showed that the wild-type strain of L. innocua is sensitive to
pediocin while the dhaR mutant is resistant. The pedB mutant is under investigation.
In addition, we purified the five proteins involved in Dha phosphorylation
and could reconstitute in vitro these metabolic steps. Interestingly the dha operon
encodes many enzymes involved in the pentose phosphate pathway especially the
Tpi-like protein. These results would suggest a link between Dha metabolism, pentose
phosphate pathway and virulence in L. monocytogenes.

AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Glucose transport system in Listeria monocytogenes and their impact
on virulence gene expression
Moussan Ake, F.
INRA, France
In Listeria monocytogenes at least two phosphoenolpyruvate (PEP): carbohydrate
phosphotransferase systems (PTS) of the mannose class and also two non-PTS
permeases can transport glucose. Mutants lacking EIIAB (Lmo0096) or EIIC
(Lmo0097) of the glucose/mannose PTS grew slightly slower on minimal medium
containing up to 10 mM glucose and consumed glucose 3-times slower than the
wild-type strain. A lmo0784 mutant (EIIA, mannose type PTS) exhibited an extended
lag phase before it started growing on glucose and glucose consumption
was slowed 3.5-fold. All PTS transporters are inactive in a ptsI mutant, which nevertheless
grew at high glucose concentrations (> 15 mM) after an extended lag
phase. Two GlcU-like non-PTS glucose transporters (Lmo0169 and Lmo0176)
probably catalyze glucose uptake in the DptsI mutant. Expression of either one of
them in an Escherichia coli DptsHIcrr mutant restored glucose utilization. The efficient
utilization of glucose affects the PrfA-dependent expression of L. monocytogenes
virulence genes, including hly. Nevertheless, inactivation of the glcU genes
in a strain carrying a Phly-gus fusion had no effect on PrfA activity. However, deletion
of the gene for EIIAB Glc/Man caused 3- to 5-fold elevated gus expression,
whereas deletion of EIIC Glc/Man had only a slight effect. b-Glucuronidase activity
in the EIIAB Glc/Man mutant was also elevated when it was grown on solid minimal
medium containing 5 or 10 mM glucose. A similar behaviour was observed for
the lmo0784 mutant, which in addition exhibits very low man operon expression.
The highest gus expression was observed in the DptsI mutant grown at glucose
concentrations between 25 and 50 mM.
Antimicrobial susceptibilities of Listeria monocytogenes isolated
in Japan
Monden, S., Okutani, A., Suzuki, H., Asakura, H., Nakama, A., Igimi, S., Okada, Y.
and Maruyama, T.
Japan
Antibiotics, such as ampicillin and co-trimoxazole, are usually used as the first
choice for treatment of listeriosis. However, the antimicrobial resistant strains of
Listeria monocytogenes are often isolated in many countries. In this study, in vitro
antimicrobial susceptibility of 232 L. monocytogenes strains (101 isolates from patients
of listeriosis and 131 isolates from food and food-related environment) obtained
in Japan from 1974 to 2008 were examined their susceptibilities against 6
kinds of antibiotics by plate dilution method. According to the breakpoint from the
guideline of National Comittee for Chemical Laboratory Standard, all strains tested
here were susceptible to gentamicin and kanamycin, and all clinical isolates
showed susceptibility to ampicillin and erythromycin. One strain from beef was resistant
to ampicillin, and the another one from pork showed resistance to 3 kinds of
drug, chloramphenicol, erythromycin and enrofloxacin. 56.4 % of clinical isolates
and 37.8 % of food and environmental isolates were resistant to enrofloxacin, and
the rest of isolates showed intermediate phenotype to this antibiotics. The rates of
enrofloxacin-resistance were very high in isolates belonging to serotype 1/2a
(63.3 %) and 1/2c (75 %) compared with those of 1/2b (41.8 %) and 4b (38 %). In this
study, most of clinical isolates were susceptible to antibiotics except enrofloxacin.
On the other hand, some strains from food and environment showed resistance or
intermediate to ampicillin, chloramphenicol or erythromycin. Furthermore, all
strains were more than intermediate to enrofloxacin, a kind of quinolone that used
for treatment of animal infection in Japan. These results suggest that the origin of
listerial isolates in involved in their antibiotic susceptibility.
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AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Influence of sub-lethal concentrations of disinfectants
on Listeria monocytogenes adhesion and invasion in Caco-2 cells
Gaedt Kastbjerg, V. 1, Halberg Larsen, M. 2, Ingmer, H. 2 and Gram, L. 1
1. National Institute of Food Research, Technical University of Denmark
2. Faculty of Life Sciences, University of Copenhagen, Denmark
Listeria monocytogenes is frequently detected in the food processing environment,
where it, ideally, must be exposed to disinfectants daily. However, L. monocytogenes
is not always eliminated by the disinfection process. One reason could be
that the bacteria are only exposed to sub-lethal concentrations of the disinfectant.
We have recently shown that sub-lethal concentrations of disinfectants used in
the food industry affect virulence gene expression on transcript level in L. monocytogenes,
and the effect depend on the active components of the disinfectants.
The aim of the present study was to determine if disinfectants used routinely in
the food industry affect the virulence of this pathogen studied in a cell-model.
Two different disinfectants were used. Compound 1 contains peracetic acid and
hydrogen peroxide as the active ingredients and reduces the expression of virulence
genes in L. monocytogenes whereas Compound 2 contains quaternary ammonium
compounds (QAC) and induces virulence gene expression. L. monocytogenes
EGD was exposed to the two disinfectants for one hour in a non-inhibiting
and a sub-lethal concentration, and subsequently the bacterial cells were added
to a monolayer of Caco-2 cells. Bacterial adhesion and invasion were monitored.
Exposure of L. monocytogenes to the sub-lethal concentration (0.0031 %) of the
QAC disinfectant significantly increased adhesion to Caco-2 cells as compared to
bacteria exposed to water (control) and resulted in a slightly higher invasion. No
effect was observed of the non-inhibiting concentration of the QAC disinfectant
(0.0016 %). In contrast, the adhesion was unaffected and the invasion slightly decreased
after exposure to the non-inhibiting concentration of 0.125 % of the peracetic
disinfectant as compared to bacteria exposed to water. On-going studies
will evaluate how long term exposure to disinfectants will affect adhesion and invasion
to Caco-2 cells and these data will also be discussed.
The SOS response in Listeria monocytogenes – a stress
survival mechanism
Kiil Nielsen, P., Zahle Andersen, A. and Haahr Kallipolitis, B.
University of Southern Denmark
The SOS response is a functionally conserved DNA repair system found in a variety
of bacteria. The SOS response involves two central regulatory components, RecA
and LexA, which coordinate the expression of target genes leading to arrest of cell
division, DNA repair and mutagenesis. Historically, the SOS response is linked to
the presence of single stranded DNA and stalled replication forks induced by UV radiation.
However several other stress conditions, which are not expected to cause
massive DNA damage, have been shown to induce SOS response e.g. mild heat
treatment, antibiotics, ethanol and salt stress. The SOS response of the food borne
pathogen Listeria monocytogenes is presently not well understood. We find it likely
that the SOS response is important for the growth and survival of this pathogen in
contaminated foods and during infection. It is therefore of great interest to investigate
this response in L. monocytogenes into more details. To this end, we focused
our studies on the auto regulated repressor protein LexA, the genes targeted by this
regulator, and the environmental signals leading to activation of the SOS response
in L. monocytogenes. LexA regulated gene expression is analyzed by comparing expression
profiles of WT L. monocytogenes with ∆lexA and lexA* strains expressing a
non functional and a constitutive active LexA, respectively. In order to further elucidate
the regulatory mechanisms and timing of the SOS response, we construct
promoter-reporter gene fusions, which report the size and duration of activation of
LexA controlled protein expression. As reporter genes we employ both lacZ and
degradation tagged gfp. We thus expect to be able to monitor time resolved changes
in SOS gene expression and thus come closer to the apparently versatile role of this
stress response mechanism in Listeria monocytogenes.

Acid shock triggers heavy metal detoxification
in Listeria monocytogenes
Müller, S., Neuhaus, K. and Scherer, S.
Technische Universität München, Germany
AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
L. monocytogenes is a food borne pathogen ubiquitous in the environment. The
bacterium is exposed to low pH in various ecological niches, such as the stomach,
macrophages or acidic soil. Acidic conditions are also found in ecological niches associated
with food production. In this work, the acid shock response at pH 5 for
15 min, 30 min, 45 min, 60 min and 120 min and the acid adaptation at pH 5.2
(growth to mid log-phase) was studied at the transcriptional level using microarrays.
Besides transcriptional induction of well characterized genes which are involved
in acid tolerance, an up regulation was also found for genes whose products
are potentially participating in heavy metal detoxification, such as ion export
proteins (heavy metal exporting ATPase: lmo0641, lmo1852-1854; cation efflux
protein: lmo2231, lmo2231) or heavy metal reduction proteins (arsenate reductase:
lmo2230). Furthermore, it was demonstrated that, after induction of the acid
tolerance response (60 min, pH 6) L. monocytogenes exhibits a higher resistance
against various heavy metal ions, e.g. against Cd II, Hg II, and Ni II; and especially
against Cu II and As V. Acidic pH appears to serve as a trigger not only for the induction
of genes involved in acid tolerance itself, but also for the induction of
genes involved in heavy metal resistance. Since metals become more bioaccessible
at low pH, especially in soil, acid solubilized heavy metals might reach noxious
doses in certain environments and, therefore, detoxification genes may be up regulated.
Ongoing analysis using deletion mutants will help to elucidate the role of
detoxification under acidic growth conditions.
Listeria monocytogenes mutants defective in growth at 5 °C
and in high salt environment
Burall, L., Laksanalamai, P. and Datta, A.
US Food and Drug Administration, USA
Listeria monocytogenes can survive and grow in refrigerated temperature and in
the presence of high salt. In an effort to better understand the associated mechanisms,
a library of 5280 transposon mutants of LS411, an isolate from the Jalisco
cheese outbreak, were screened for their ability to grow in brain heart infusion
(BHI) broth at 5 °C or in the presence of 7 % NaCl. Two mutants with altered
growth profiles have been identified. 63-C8 showed a significant reduction in
growth in BHI at 5 °C. Additionally, the mutant had reduced growth in the presence
of 9 % but not 7 % NaCl. Sequencing located the transposon between two divergently
transcribed genes, iap and one encoding a putative SecA subunit. qPCR
revealed a substantial reduction in the expression of iap in 63-C8 relative to LS411,
though a slight increase in expression was observed for the putative SecA subunit
gene. 36-F11 showed no growth reduction at 5 °C but had attenuated growth in the
presence of 7 % NaCl. The salt attenuation was exacerbated at 9 % NaCl, which resulted
in reduced colony counts despite apparent increases in biomass. Preliminary
microscopy work showed that the majority of cells in these cultures are “live”,
indicating the possible presence of either a viable but non-culturable state or increased
avidity in the bacterial associations within the clumps. Sequencing of the
insertion region of 36-F11 identified the transposon between two divergently transcribed
genes. The insertion site is 36bp upstream of a gene encoding a glycine
betaine/L-proline ABC transporter, which is down-regulated in the mutant while
the divergently transcribed putative mechanosensitive ion channel appeared
slightly upregulated. Further studies are being conducted to identify the mechanisms
underlying these phenotypes.
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AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Revival of 5,000 Listeria strains from Seeliger’s historical collection
with a semi-automated microbiological pipeline
Haase, J. 1, Hof, H. 2 and Achtman, M. 1
1. Science Foundation Ireland
2. University of Heidelberg, Mannheim, Germany
Prof. Seeliger from the University of Würzburg, Germany, collected more than
7,000 Listeria strains, globally, between the 1920s and the 1980s from humans, animals,
food and the environment. Within this project, this collection, the “Special
Listeria Culture Collection” (SLCC), has been recultured and frozen at -80 °C.
Strain information has been transcribed from the original notebooks and is now
stored in an SQL-based database (Bionumerics, Applied Maths) and the physical
strain locations are stored in a LIM system (ItemTracker). A large collection such
as this required the set up of a comprehensive “strain pipeline”. This ensures correct
cataloguing, efficient storage and retrieval of cultures. This was achieved via
custom scripts (Python) that allow communication and archiving of data obtained
from the initial culturing to isolation of DNA and downstream phylogenetic analyses.
The collection now provides an exceptional opportunity to investigate the
population structure and the evolutionary history of L. monocytogenes.
Two point mutations are responsible for the lack of glycosidic substition
in cell wall teichoic acids in Listeria monocytogenes serovar “7”
Eugster, M. R. 1, Huwiler, S. 2, Morax, L. 1 and Loessner, M. J. 1
1. Institute of Food, Nutrition and Health, ETH Zurich, Switzerland
2. Institute of Microbiology, ETH Zurich, Switzerland
The currently defined six Listeria species are further differentiated into at least 15
distinct serovars based on serological reactions with specific antisera directed
against O and, to a lesser extent, H antigens. Some L. monocytogenes strains (such
as WSLC 1034) have previously been assigned to serovar “7”. These cells contain
ribitol phosphate wall teichoic acids (WTA) similar to serovars 1/2 and 3, but without
glycosyl substition. Purified WTA polymers and associated sugars were analyzed
by a novel technique using electrospray ionization tandem mass spectrometry
(ESI-MS/MS). In addition, WTA carbohydrates were identified using
fluorescently labeled N-acetylglucosamine (GlcNAc) binding proteins (CBD-P35)
and Listeria phage A118 which requires rhamnose-substituted WTAs for adsorption.
We found that the absence of rhamnose and GlcNAc in WTAs of strain 1034 is
based on two single point mutations only. The loss of GlcNac is due to a mutation
(P300L) in the protein of gene lmo2550, which is involved in wall teichoic acid
decoration with GlcNAc. The second mutation is a single base deletion and produces
a frameshift at position 165 in gene lmo1083, whose product is involved in
rhamnose biosynthesis, resulting in a truncated enzyme and lack of rhamnose in
WTA. Deletion mutants constructed in a serovar 1/2a background (EGDe
Dlmo2550 Dlmo1083) resulted in loss of the rhamnose and GlcNAc WTA components.
Complementation with wt lmo2550 and lmo1083 in the EGDe mutant, in
serovar “7” strain 1034, and in other mutants featuring a lack of sugar substitution
not only restored the rhamnose and GlcNAc content in EGDe, but also produced
a serovar 1/2 phenotype in the “serovar 7” strain and other mutants lacking
GlcNAs from their WTA. These findings suggest that Listeria serovar “7” strains (at
least WSLC 1034) are likely mutants originating from a serotype 1/2 background,
and that Listeria monocytogenes serovar “7” may not exist.

AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
High-throughput genome sequencing of two Listeria monocytogenes
clinical isolates during a large foodborne outbreak
Gilmour, M. 1, Graham, M. 1, Van Domselaar, G. 1, Tyler, S. 1, Kent, H. 1, Trout-Yakel, K. 1,
Larios, O. 1, Allen, V. 2, Lee, B. 3, Nadon, C. 1 and Kearney, A. 1
1. Public Health Agency of Canada
2. Ontario Agency for Health Protection and Promotion, Canada
3. Canadian Food Inspection Agency, Canada
A large, multi-province outbreak of listeriosis associated with ready-to-eat meat
products contaminated with Listeria monocytogenes serotype 1/2a occurred in
Canada in 2008. Subtyping of outbreak-associated isolates using pulsed-field gel
electrophoresis (PFGE) revealed two similar but distinct AscI PFGE patterns.
Whole genome sequencing of two L. monocytogenes isolates was completed using
the Roche GS FLX platform to rapidly determine the genome sequence of the
primary outbreak strain and to investigate the genetic diversity associated with a
change of a single restriction enzyme fragment observed by PFGE. The chromosomes
were collinear, but differences included 28 single nucleotide polymorphisms
(SNPs) and three indels, including a 33 kbp prophage that accounted for
the difference in PFGE pattern. The distribution of these traits was assessed
within clinical, environmental and food isolates associated with the outbreak, and
this comparison indicated that three distinct, but highly related strains may have
been involved in this nationwide outbreak. Notably, these two isolates were found
to harbor a 50 kbp putative mobile genomic island encoding translocation and efflux
functions that has not been observed in other Listeria genomes. Highthroughput
genome sequencing therefore provided a more detailed real-time assessment
of genetic traits characteristic of the outbreak strains than could be
achieved with routine subtyping methods. These data allowed us to determine
evolutionary lineages and unequivocally define the full breadth of genetic variation
between two subtype variants identified by the internationally standardized
PulseNet PFGE typing method. This study confirms that the latest generation of
DNA sequencing technologies can be applied during high priority public health
events, and laboratories need to prepare for this inevitability and assess how to
properly analyze and interpret whole genome sequences in the context of molecular
epidemiology.
Expression of antimicrobial activity in food and clinical
Listeria monocytogenes isolates
Barbosa, J., Ferreira, V., Borges, S., Azevedo, I., Magalhães, R., Santos, I, Almeida, G.
and Teixeira, P.
Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Portugal
Listeria monocytogenes is a Gram-positive facultative intracellular pathogen capable
of causing fatal infections in risk groups, such as pregnant women, elderly and
immunocompromised individuals. Little information is available on the antimicrobial
susceptibility of L. monocytogenes, particularly of strains isolated from food
and production environments. Minimum inhibitory concentrations (MIC’s) of
ampicillin, penicillin G, chloramphenicol, erythromycin, tetracycline and vancomycin
were assessed for 370 and 118 food and clinical L. monocytogenes isolates,
respectively. All the clinical isolates were susceptible to all the antibiotics tested.
Amongst food isolates, 0.5 % were resistant to tetracycline and 4.1 % and 2.7 % were
intermediary and resistant, respectively, to erythromycin. Only one isolate demonstrated
a resistance profile to tetracycline, an antibiotic largely used in veterinary
practice, but the resistance of nine isolates to erythromycin is a reason for concern,
since erythromycin is used to treat pregnant women diagnosed with listeriosis or as
a drug of second choice in cases of ampicillin allergy. Although in vitro antibiotic
susceptibility testing does not always reflect the in vivo situation, results demonstrated
that some of the isolates investigated are resistant to antibiotics of clinical
importance commonly used to treat listeriosis. Listeriosis is usually an easily treatable
disease and no reports have been presented on antibiotic resistant isolates
from disease cases. However, it is an important area to keep under surveillance.
Whilst the present study does not in a scientific sense present de novo science, it
contains very valuable and important data. Only by keeping an on-going track of
the resistance patterns can these be monitored for unexpected changes.
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AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
The Listeria monocytogenes sigma B and sigma H regulons overlap, but
only sigma B appears to be important for survival of acid, alkaline and
oxidative stress
Chaturongakul, S. 1, Raengpradub, S. 2, Wiedmann, M. 3 and Boor, K. J. 3
1. Mahidol University, Thailand
2. Silliker, Inc., USA
3. Cornell University, USA
Previous transcriptomic analyses in Listeria monocytogenes using whole genome microarrays
and Hidden Markov Model (HMM) based promoter searches identified
many genes that were controlled by more than one transcriptional regulator (e.g., σ B,
σ H, σ L, PrfA, HrcA or CtsR). The regulons of the two stationary phase sigma factors,
σ B and σ H shared the largest number of genes. To further investigate the regulatory
features shared between σ B and σ H, whole genome microarrays were performed and
transcript profiles for ∆sigB, ∆sigH, and ∆sigB∆sigH strains were compared to that of
the parent strain 10403S. In the present study, using cut-off criteria of a 1.5 fold difference
at p < 0.05, we identified 301 σ B-dependent genes, 82 s H-dependent genes,
and a total of 61 genes with expression profiles that suggest regulatory interactions
between σ B and σ H. The 61 co-regulated genes grouped into 4 different quality
threshold (QT) clusters: Cluster 1 is comprised of 19 genes that are positively regulated
by both σ B and σ H (e.g., mRNA levels for cspD, encoding a cold shock protein
and for rpoD, encoding an RNA polymerase sigma factor, were significantly lower in
the ∆sigB∆sigH strain than in the other strains), Cluster 2 contains 17 genes that are
negatively regulated by both σ B and σ H (e.g., mRNA levels for genes encoding ribosomal
proteins (rpsR and rplJ), RNA polymerase beta subunit (rpoB), and a sigma factor
(sigC) were significantly higher in the ∆sigB∆sigH strain than in the other strains),
Cluster 3 contains 11 genes with reduced expression in both the ∆sigB and ∆sigH
strains, but with transcript levels that are the same in both the ∆sigB∆sigH and wild
type strain (e.g., ATP-dependent Clp protease clpP), and Cluster 4 has 14 genes that
are positively regulated in the ∆sigB strain but that are negatively regulated in the
∆sigB∆sigH strain or vice versa (e.g., mreD, which contributes to cell shape and rpsD
and rplK, which encode ribosomal proteins). In addition to transcript profiling,
10403S and the 3 otherwise isogenic mutant strains were also phenotypically characterized.
After exposure to acid (1 h at final pH of 2.5), alkaline (1 h at final pH of 12), or
oxidative stresses (15 min at 13 mM cumene hydroperoxide final concentration), the
phenotype of the ∆sigB∆sigH strain was identical to that of the ∆sigB strain, i.e., with
cell numbers that were significantly reduced (CFU/ml) when compared to those of
the parent strain. This latter finding further verifies the central role of σ B in stress response,
and suggests that σ H is less important for survival of these stresses.
Virulence gene expression in Listeria monocytogenes strains isolated
from different sources
Alessandria, V., Rantsiou, K. and Cocolin, L.
University of Turin, Italy
Listeria monocytogenes is an important food-borne pathogen that has the capacity to
cause severe infections. Ubiquitous microorganism, it is commonly isolated from
foods of animal origin, mainly meat and milk products. However, due to its capacity to
develop at refrigeration temperatures, human listeriosis outbreaks are most often associated
with ready-to-eat food products that are consumed without prior cooking.
The incidence of the disease depends on different factors including the infective dose
and the immunity conditions of the host. The present work focuses on the expression
analysis of four virulence genes (sigB, plcA, hly and iap) in 11 different strains of L.
monocytogenes and, more in detail, 3 collection strains (EGDe, NCTC10527, SCOTT
A), 7 isolated from food matrices (4 of which isolated from meat products and three
from diary products) and 1 isolated from humans. In the first step, the expression
analysis was performed in vitro, culturing each strain in BHI (Brain Heart Infusion)
medium, in order to identify possible differences in the expression level for the different
strains. The combined use of reverse transcription and quantitative PCR (qRT-
PCR) was used to evaluate gene expression. As a second step, we wanted to evaluate
the trend of expression of the genes in food. Analyses were performed inoculating L.
monocytogenes in milk at different conditions and in particular at two temperatures
(4 and 12 °C) for different times (24 and 48 hours). Significant expression differences
emerged for the different genes, without showing any significant association between
the expression of the genes and the origin of the different strains.

AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Differentiation of Listeria monocytogenes, Listeria innocua and
Listeria marthii, a novel Listeria species isolated from the natural
environment, Finger Lakes National Forest
Graves, L. M. 1, Helsel, L. O. 1, Steigerwalt, A. G. 1, Morey, R. E. 1, Daneshvar, M. I. 1,
Roof, S. E., 2 Orsi, R. H. 2, Fortes, E. D. 2, Milillo, S. R. 2, den Bakker, H. C. 2, Wiedmann, M. 2,
Swaminathan, B. 1 and Sauders, B. D. 3
1. Centers for Disease Control and Prevention, USA
2. Cornell University, USA
3. New York State Department of Agriculture and Markets Food Laboratory Division, USA
Four isolates (FSL S4-120 T, FSL S4-696, FSL S4-710, and FSL S4-965) of Grampositive,
motile, facultatively anaerobic, non-sporeforming bacilli that were phenotypically
similar to Listeria spp. were isolated from soil, standing water, and flowing
water samples obtained from the natural environment in the Finger Lakes
National Forest, New York, USA. The four isolates were closely related to one another
and were determined the same species by whole genome DNA-DNA hybridization
studies, but sufficiently different from L. monocytogenes and L. innocua
by DNA-DNA hybridization to form a separate species. 16S ribosomal RNA sequence
analysis confirmed their close phylogenetic relatedness to L. monocytogenes
and L. innocua and more distant relatedness to L. welshimeri, L. seeligeri, L. ivanovii,
and L. grayi. Phylogenetic analysis of partial sequences for sigB, gap, and prs
showed that these isolates form a well-supported sistergroup to L. monocytogenes.
The four isolates showed > 82 % relatedness at 55 °C and > 76 % relatedness at 70 °C
with 0.0 – 0.5 % divergence. Labeled DNA from the type strain (FSL S4-120 T)
showed an average of 73 % relatedness to three L. monocytogenes strains and one L.
innocua strain (range, 69 – 75 %) in reactions at 55 °C, the divergence in related DNA
sequences was between 7.5 % and 9.5 %. In reactions at 70 °C, FSL S4-120 T DNA
showed an average of 45 % relatedness to the three L. monocytogenes strains and
one L. innocua strain (range, 30 – 53 %). The isolates yielded positive reactions in
the AccuProbe® test that is purported specific for L. monocytogenes, did not ferment
L-rhamnose, were non-hemolytic on blood agar media, and did not contain a
homologue of the L. monocytogenes virulence gene island. A new species L. marthii
is described which phenotypically and genotypically is distinct from all other Listeria
species including its closest neighbors L. monocytogenes and L. innocua.
Differed roles of L,D-carboxypeptidases encoded by lmo0028
and lmo1638 genes.
Yurov, D.*, Varfolomeev, A., Kaminskaya, A. and Ermolaeva, S.
Gamaleya Institute of Epidemiology and Microbiology, Moscow, Russia
L,D-carboxypeptidase hydrolyses a bound between m-A 2 pm and C-terminal Dalanine
in murein. Listeria monocytogenes carried two genes, lmo0028 and
lmo1638 that encode L,D-carboxypeptidases. The lmo0028 promoter has a recognition
site for the virulence regulator PrfA. Mutant L. monocytogenes strains
GIM0028 and GIM1638 were obtained by site-specific insertions into the chromosome
of the wild type EGDe strain. Morphology was characterized with light
microscopy. Septation was studied with fluorescent vancomycin. Genome copy
numbers were determined with quantitative PCR. L. monocytogenes virulence was
studied on BALB/c mice and human colon carcinoma HT29 cells. The mutation
in lmo0028 caused dispersion in cell lengths: the length/width ratio ranged between
1.5 and 15 for GIM0028 and 1 and 4.4 for EGDe. The insertion in lmo1638
caused shortening in cells. The average length/width ratios were 3.75, 1.2 and 3.0
for GIM0028, GIM1638 and EGDe, respectively. The longish GIM0028 cells were
defected in septum formation, GIM1638 were not. GIM0028 demonstrated two
fold decreasing in invasion efficiency. Doubling times in HT29 cells determined by
plating were 51 minutes and 70 minutes for EGDe and GIM0028, respectively.
However, evaluation of intracellular bacterial genome numbers with qPCR did not
reveal a difference between the strains. GIM0028 bacteria increased their length
upon intracellular growth: average length/width ratio was about 4.8 for invading
bacteria and 9.2 after 6 h of intracellular growth while for the wild type bacteria it
was 3.5 (p < 0.005). GIM0028 demonstrated reduced virulence for BALB/c mice
with LD 50 of 1x10 6 and 1.5x10 4 CFU/mouse for GIM008 and EGDe strains, respectively.
The Lmo0028 L,D-carboxypeptidase is involved in septum formation
while Lmo1638 seems to be involved in cell elongation. A mutation in lmo0028
decreases L. monocytogenes virulence.
* Participation Supported by IUFoST.
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AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Antimicrobial susceptibilities of Listeria monocytogenes isolated
from retail beef, pork and poultry in Japan
Ida, M., Shimojima, Y., Kaneko, S., Higuchi, Y., Nakama, A. and Kai, A.
Tokyo Metropolitan Institute of Public Health, Japan
Meat and meat products are the major source of food-borne infections and the
most important link between food-producing animals and humans. The aim of
this study is evaluate antimicrobial susceptibilities of Listeria monocytogenes isolated
from meat in Japan. A total of 125 strains isolated from beef (n = 10), pork
(n = 49) and poultry (n = 66) between 2001 and 2009 in Tokyo area were examined.
Serotypes of tested strains were 1/2a (n = 46), 1/2b (n = 26), 1/2c (n = 15), 4b (n = 26)
and others (n = 12, including untypable). Ampicillin, penicillin G, gentamicin,
kanamycin, erythromycin, oxytetracycline, norfloxacin and chloramphenicol were
included in the study. The minimum inhibitory concentrations of antimicrobial
agents were determined by the agar dilution method according to the guidelines of
Clinical Laboratory Standards Institutes (CLSI). CLSI breakpoint guidelines for L.
monocytogenes and other Gram-positive bacteria were used for data analysis. Enterococcus
faecalis ATCC 29212 and Escherichia coli ATCC 25922 were used as
quality control strains. All isolates were susceptible to antimicrobials except for
oxytetracycline. Three strains, isolated from domestic pork, domestic poultry and
beef imported from Australia were resistant (> 32µg/ml). All of them were isolated
in 2001 and belonging to serotype 1/2c. This study showed that antimicrobial resistance
is not highly prevalent in L. monocytogenes in meat in Japan.
Genetic basis of two low pathogenic L. monocytogenes strains
with apparent phospholipase C activity
Jiang, L., Bai, F., Chen, J., and Fang, W.
Zhejiang University Institute of Preventive Veterinary Medicine, Hangzhou, China
Two Listeria monocytogenes isolates showing apparent in vitro phospholipase C
activity were characterized for their genetic basis and pathogenecity. Sequencing
analysis found two major mutations in plcB at position 1 [from A (ATG) to G
(GTG)] and position -26 [from C (ACG) to T(ATG)], resulting in 9-aa extension of
the enzyme at the N-terminus. The strains were of serovar 4a, had low pathogenecity
with LD 50 at log 10 CFU 8.21-8.35 in BALB/c mice, and presented no microscopically
visible plaques on the L929 cell monolayers. Site-directed mutation
of these sites to the plcB version of strain 10403S abolished the enzyme activity, increased
virulence by one log (from 8.12 to 7.07), but did not have significant effect
on plaquing. This abolishment did not seem to be related to presence of G145S in
the central regulator prfA, which was found to induce overexpression of virulence
genes independent of environmental conditions in other strains. However, this
does not mean that prfA was not involved in regulation of its expression because
transposon mutagenesis identified two mutant strains lacking apparent phospholipase
activity due to disruption of the prfA by insertions at nt364 and nt828. There
are two additional changes of T165A and K197N in prfA, as compared with other L.
monocytogenes serovar 1/2a and 4b strains. Further work is under way to examine
if apparent activity of the novel phospholipase in these strains results from easy
access to zinc metalloprotease encoded by mpl for maturation, or if amino acid
substitutions of T165A and K197N other than G145S in prfA also contribute to regulation
of downstream virulence genes. Using one of the strains as a model organism,
we are testing how phospholipase C and listerolysin interact in determining
its pathogenecity and if there are other genes involved in their low pathogenecity
by genome wide scanning using Solexa sequencing technology.

AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Molecular genotyping and antimicrobial resistance
of Listeria monocytogenes from foods and the environment
Parisi, A. 1, Miccolupo, A. 1, Fraccalvieri, R. 1, Latorre, L. 1, Normanno, G. 2 and Santagada, G. 1
1. Experimental Zooprophylactic Institute Apulia and Basilicata, Italy
2. University of Bari, Faculty of Veterinary Medicine, Italy
Listeria monocytogenes is generally susceptible to a wide range of antibiotics, however
an increasing number of strains resistant to one or more antibiotics have been
reported. Objectives of the present study were to evaluate molecular genotyping
and antimicrobial susceptibility in L. monocytogenes isolates from foods and the
environment. Over all a total of 100 L. monocytogenes isolates from foods (n = 74)
and the environment (n = 26) were subjected to molecular genotyping using Multi-
Locus Sequence Typing as previously described, moreover the susceptibility to 21
antimicrobials was performed using Gram-positive GPN4F kit (TREK Diagnostic
System). All the isolates resulted sensitive to ampicillin, chloramphenicol, gentamycin,
penicillin G, rifampicin, streptomycin, sulfamethoxazole trimethoprim.
Antimicrobial resistance was recorded for oxacyllin (24 %), and tetracycline (1 %)
whereas some isolates resulted moderately sensitive toward clindamycin (19 %),
quinupristin/dalfopristin (2 %) and ciprofloxacin (1 %). The isolates belonged to
six different serotypes: 1/2a (43 %), 1/2c (24 %), 1/2b (15 %), 4b/4e (12 %), 3a (4 %)
and 3b (2 %). MLST identified 50 different Sequence Types (ST) most of which
represented by a single isolate. ST9 (21 %), ST121 (12 %), ST3 (5 %) and ST199 (5 %)
were the most prevalent STs. Moreover MLST allowed a good discrimination of
the two main genetic lineage of L. monocytogenes. Our results confirm the low
prevalence of food and environmental isolates resistant to antimicrobials. Interestingly,
a good correlation between resistance to oxacillin and genetic lineage I
was observed (Yates corrected = 35,46; p < 0.005); this is in agreement with the
clonal organization of L. monocytogenes population. The severity of L. monocytogenes
infections and the reported increasing of antimicrobial resistance make necessary
the continuous monitoring of this trend although the recorded data do not
appear to be alarming. The comparison of phenotypic and genetic characters allow
to validate the evolutionary models speculated for this microbial species.
Virulence transcriptome analysis of Listeria monocytogenes
by application of microarrays in vitro and in situ
Rantsiou, K., Alessandria, V. and Cocolin, L.
University of Turin, Italy
With the complete genome sequence available for Listeria monocytogenes as well
as other Listeria spp., it is nowadays possible to use tools that allow global analysis
of the biology of this pathogenic microorganism. Microarrays give the possibility to
study concurrently a large number of genes and deduce information regarding
their expression. This approach accelerates significantly the rhythm by which new
information is generated. In this way, the effect of different conditions on the expression
of a series of genes and as a consequence on the physiology of a microorganism
can be investigated. Within the 6 th FP project Pathogen Combat, it was
developed a microarray including 72 genes of L. monocytogenes, encoding for virulence,
adhesion and stress response genes. In this study we sought to apply this
array to study the effect of different environmental conditions on the expression of
virulence genes. First, different strains of L. monocytogenes were grown in vitro, at
different temperatures, pH and salt concentrations. Then, L. monocytogenes was
artificially inoculated in different food matrices, to simulate the conditions of ‘virulence’
of this microorganism at time of consumption. Global analysis of the expression
of virulence genes in different L. monocytogenes strains will lead to a better
undestanding of the physiology of this microorganism and eventually to the
prediction of its potential to cause disease to humans.
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AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Effects of growth conditions on surface properties of Listeria;
a proposed role for AI-2
Wong, H. T. L., Nwaiwu, O.* and Rees, C. E. D.
School of Biosciences, University of Nottingham, UK
Listeria monocytogenes is recognized as a particular problem in factory environments
due to its ability to colonize surfaces and then become persistent by adapting
to low nutrient and low temperature conditions. We have been investigating
the effect of low nutrient conditions on the ability of Listeria to attach to surfaces.
Cultures were prepared using a minimal medium (D10), a rich medium (BHI) and
also the D10 medium supplemented with duck meat extract. Growth in the BHI
medium was fastest, but significant increases in growth were seen when D10 was
supplemented with 10 % duck meat extract. One feature of cells that become persistent
in factory environments is their ability to form biofilms that are resistant to
cleaning and disinfection. We have found that the surface hydrophobicity (measured
using Microbial Attachment to Solvents (MATS) assays) alters significantly
when the organism is grown under low nutrient conditions, with cells becoming
more hydrophobic. Also these cells have a greater tendency to flocculate in liquid
culture. This affect is seen when cells are grown above 30 °C and therefore is not
related to the synthesis of flagellae. The ability to form biofilms in Listeria is
known to be affected by the synthesis of a functional autoinducer 2 (AI-2)-like
signal, and that an intact luxS gene is associated with inhibition of attachment and
biofilm formation. Using a Vibrio harveyi reporter strain we have found that when
cells are grown in minimal media, the amount of detectable AI-2 is reduced. As
AI-2 has a role in the down-regulation of components required for attachment
and biofilm formation, this result is consistent with our observation of changes
in the surface properties of Listeria under low nutrient conditions that are more
likely to promote surface attachment.
* Participation Supported by IUFoST.
Stress behaviour of a Listeria monocytogenes 568 Lmo1634
transposon mutant
Truelstrup-Hansen, L. 1, Holman, D. B. 1 and Ells, T. C. 2
1. Dalhousie University, Canada
2. Agriculture and Agri-Food Canada
Listeria monocytogenes is an important psychrotrophic foodborne pathogenic bacterium
with tendency to persist in food processing plants. Heat treatment is routinely
used as a means to control pathogenic bacteria in food products. Therefore,
the thermotolerance of L. monocytogenes and mechanisms responsible are of interest.
Previous research using transposon mutagenesis yielded several heat resistant
mutants, including one, named 1B4, that was mapped to a putative alcohol/acetaldehyde
dehydrogenase gene, lmo1634 or adh, also identified as Listeria adhesion
protein. The objective of this study was to investigate the function of this gene
in relation to a variety of environment stresses and in biofilm formation. The transposon
insertional mutant 1B4 demonstrated significantly greater tolerance to acid
(pH 2.7), 20 % NaCl, and heat treatment of 52 °C than the wild-type (WT) Lm568
strain. Attachment of 1B4 (7.06 log 10 CFU/cm 2) to stainless steel coupons was not
significantly (p > 0.05) different from the WT Lm568 (6.91 log 10 CFU/cm 2). Neither
1B4 nor Lm568 exhibited greater survival after desiccation (43 % RH) at 20 °C for 7
days. The alcohol dehydrogenase activity of Lm568 (0.197 U/mg) was found to be
significantly (p > 0.05) greater than in 1B4 (0.0458 U/mg). 1B4 was complemented
in trans with the WT lmo1634 gene and the successful transcription of lmo1634 in
the complemented strain was confirmed by RT-PCR. In addition, no polar effect on
the genes immediately downstream of lmo1634 in 1B4, i.e., lmo1635, lmo1636, or
lmo1637, was observed. The trans complementation of 1B4, however, did not completely
restore the WT phenotype. Attempts to create a deletion mutant in lmo1634
ultimately proved unsuccessful. Despite these technical issues, it is clear that a mutation
in lmo1634 yields a multi-stress resistant phenotype.

AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Investigation of the conditions that trigger the activation of the alternative
sigma factor σB in Listeria monocytogenes
Utratna, M., Shaw, I. and O’Byrne, C.
National University of Ireland, Galway, Ireland
Listeria monocytogenes has evolved multiple strategies to overcome diverse stress
conditions and it is able to occupy extreme environmental niches. As a facultative
intracellular foodborne pathogen L. monocytogenes successfully invades host cells,
surviving in gastric tract before the final colonization of the target organs. The alternative
sigma factor (σ B) plays an essential role in the stress tolerance and virulence
of L. monocytogenes. Many components of the σ B regulon have been identified
but the mechanisms that regulate σ B are still unknown. The current model
for σ B regulation, based on work done in Bacillus subtilis, suggests that the activity
of σ B is independent of the levels of σ B in the cell. The expression of OpuCA has
been shown to be controlled by σ B. Therefore the levels of this protein can act as an
indirect reporter of the activity of σ B in L. monocytogenes. Polyclonal antibodies
against OpuCA were developed in chickens and used in Western blotting. Based
on this reporter system it was shown that σ B is more active in stationary phase
when compared to exponential phase (OD 600 =0.6). σ B activity under conditions
encountered in the human gut was also determined. Levels of OpuCA increase
gradually in the range of salt up to 0.9M NaCl and when the pH is reduced from pH
7.2 to pH 5.0. Higher σ B activity was also detected under limited oxygen conditions
and at 4 °C. However, no change in the levels of OpuCA was observed in the
presence of low concentrations of bile salts up to 5mM. Additionally an attempt
was made to develop a transcriptional reporter fusion system by cloning the σ B
promoter from strongly σ B-dependent gene, lmo2230, into the reporter vector
pTCV-lac allowing the measurement of β-galasctosidase activity under various
conditions. This genetic system will also be a useful tool in monitoring the activity
of σ B in L. monocytogenes.
Characterization of Listeria monocytogenes 1/2a, 1/2b, 1/2c and 4b
by Amplified Fragment Length Polymorphism and evaluation of their
geographical distributions in Portugal
Maia, C. H. 1, Goulão, M. M. 2, Santos, M. I. 1, Ferreira, M. A. S. S. 3 and Pintado, C. M. B. S. 2
1. Instituto Nacional de Saúde Doutor Ricardo Jorge, Portugal
2. Escola Superior Agrária, Instituto Politécnico de Castelo Branco, Portugal
3. Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Portugal
Listeriosis is a bacterial infection caused by the ingestion of foods contaminated
with Listeria monocytogenes, that can affect humans, especially the group that includes
pregnant women, newborn infants, the elderly and immunocompromised
individuals. AFLP molecular subtyping, using EcoRI restriction enzyme, was used
to do the differentiation between strains isolated from different Denomination of
Protected Origin regions of cheese production in Portugal and as well from clinical
specimens. A set of 159 strains belonging to serotypes 1/2a, 1/2b, 1/2c and 4b isolated
from samples of Serra da Estrela ewe’s cheese, São Jorge cow´s cheese, Serpa
ewe´s cheese, Tolosa ewe´s cheese and Castelo Branco ewe´s cheese were analyzed.
Evaluation of similarities between strains was obtained by analyses with BioNumerics
software by Applied Maths, Belgium. With AFLP molecular subtyping,
nearly all the isolates were divided according to their serovar. Therefore it was
possible to predict the serovar from the AFLP pattern found in most of the isolates.
In the opposite direction, each one of the serovars was associated to several
AFLP molecular types, which demonstrates the grater discriminatory capacity of
the AFLP subtyping. Similarities between profiles, based on band positions, were
derived from the Dice correlation coefficient (S D ) with a maximum position tolerance
of 1 %, 3 % or 4 %. Listeria monocytogenes strains were clustered by the technique
of the UPGMA and a dendrogram was constructed to reflect the genetic distance
between them.
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AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Global analysis of the Listeria monocytogenes surface proteins
of the LPXTG family
Botello-Morte, L. 1, Calvo, E. 2, Mariscotti, J. 1, D’Orazio, V. 1, García-del Portillo, F. 1
and Pucciarelli, M. G. 1,3
1. Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior
de Investigaciones Científicas (CSIC). Darwin 3, Madrid, Spain
2. Unidad de Proteómica, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
3. Departamento de Biología Molecular, Universidad Autónoma de Madrid. Campus de Cantoblanco,
Madrid, Spain
Listeria monocytogenes is a Gram-positive intracellular bacterial pathogen that
causes serious systemic diseases in humans and animals. Comparative genomics
revealed the presence in the Listeria genus of a large number of genes encoding
surface proteins containing an LPXTG sorting motif. This signature makes these
proteins recognized by sortases, the enzymatic machinery that anchors proteins
covalently to the peptidoglycan. In the case of the L. monocytogenes EGD-e strain,
a total of 41 genes encoding LPXTG proteins were discovered. Despite the relevance
of surface proteins for communication with the environment and the host,
the biological role of most members of this family remains unknown in L. monocytogenes.
In a collaborative effort with other European groups that contributed to
generate mutants defective in specific LPXTG proteins, we accomplished a comprehensive
proteomic analysis in peptidoglycan material purified from each of
these mutants. A total of 30 LPXTG mutants have been analysed to date. Proteomic
analysis was also performed in peptidoglycan purified from intracellular
wild-type bacteria upon infection of epithelial cells. A major finding of this global
approach was the cross-regulation existing among certain LPXTG proteins. This
phenomenon involved in most cases the down-regulation of concrete LPXTG proteins
in response to the lack of a specific LPXTG protein. Interestingly, the inverse
phenomenon, the up-regulation of a LPXTG protein in response to a deficiency
in another LPXTG protein, was also observed. These proteomic data were confirmed
with specific antibodies. Proteins subjected to these regulatory processes,
currently investigated in detail by our group, include Lmo0320 (Vip), Lmo0514,
Lmo0610, Lmo0880, InlE, InlG, InlH, and Lmo2085.
Antimicrobial susceptibility of Listeria monocytogenes strains
derived from food and food-processing bakery plant
Eusébio, C., Carneiro, L., Santos, I., Magalhães, R., Almeida, G., Silva, J. and Teixeira, P.
CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal
The susceptibility of 167 strains of Listeria monocytogenes isolated from a bakery
industry, from food and food-processing environment, to 11 antibiotics was determined
by the standard agar dilution methodology. The tested antibiotics were:
ampicillin, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, nitrofurantoin,
penicillin, rifampicin, streptomycin, tetracycline and vancomycin; minimal
inhibitory concentrations values were used to classify the strains into sensitive,
moderately resistance and resistant. All the tested isolates were found to be
susceptible to ampicillin, chloramphenicol, gentamicin, nitrofurantoin, penicillin,
rifampicin, tetracycline streptomycin and vancomycin. In the case of erythromycin,
54 isolates (32 %) were susceptible, 68 (41 %) displayed moderately resistance
and 45 (27 %) were resistance. Concerning to ciprofloxacin, a moderate resistance
was observed in 12 strains (7 %) against 155 strains (93 %) that were susceptible.
Generally, this study showed that L. monocytogenes strains are susceptible to the
antibiotics commonly used in the treatment of listeriosis. Concerning that antibiotic
resistance in some L. monocytogenes strains has already been described, a continued
study of emerging antimicrobial resistance is important to guarantee an
effective treatment of human listeriosis.

A physiological study to purpose a new formal method
to obtain L. monocytogenes cells adapted to BAC
AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATIONS // P / 00–46
Saá Ibusquiza, P., Cabo, M. L., Herrera, J. J. R., Vázquez, D., Carrera, S. and Eiriz, E.
Instituto de Investigaciones Marinas (Marine Research Institute), CSIC, Vigo, Galicia, España
Maximum adaptation potential of Listeria monocytogenes (L. monocytogenes) cells
to benzalkonium chloride (BAC) was classically checked by successive exposition
of stationary cells to increasing sublethal BAC concentrations. However, this
method is time consuming and do not ensure we reach the maximum level of adaptation
of the strain. So, in the present work we develop a new method to obtain
BAC-adapted cells of L. monocytogenes that is based in only one exposition of
L. monocytogenes exponential-phase cells to sublethal concentrations of BAC.
Moreover, in order to predict the optimal conditions for achieving the maximum
level of adaptation, a factorial design to formalize the effects of the initial concentration
of cells and the concentration of BAC during the exposition was carried
out. Obtained empirical model demonstrated a positive effect of inoculum and a
positive interaction between both variables. However, a negative first order effect
for the BAC indicated the convenience of using moderately high concentrations of
it during the expositions. As a consequence of applying this procedure, adaptation
of L. monocytogenes 5873 to BAC was increased aproximately 4 times respecting to
the wild type strain in only 36 h. Finally, comparison between the expression profiles
in the wild and the highest BAC-adapted variant was also carried out.
Characterization of a RNA-helicase in the human pathogen
Listeria monocytogenes
Netterling, S. and Johansson, J.
Department of Molecular Biology, Umeå University, Umeå, Sweden
The intracellular pathogen Listeria monocytogenes grows in a wide range of milieus
and shows a great acceptance to different environmental conditions, such as
being able to grow in a span from -1 to 45 °C. Listeriosis can cause severe symptoms
such as meningitis, with high lethality rate. The ability to grow at low temperatures
is one of the most interesting aspects of this pathogen. We have looked at
RNA helicases; proteins that are able to unwind short dsRNA structures in an ATPdependent
manner. Most mRNAs develop complex secondary and tertiary structures
during the maturation process, these structures needs to be unwound before
translation initiation. The stability of these complex structures is influenced
by temperature, often becoming more rigid at lower temperatures thereby hindering
e.g. translation, resulting in undesired cold-stabilized RNA structures. RNA
helicases may function by relaxing local RNA-RNA interactions or by dissociating
RNA from proteins. In L. monocytogenes RNA helicases have earlier been
shown to be up-regulated during growth in a cold environment. RNA-helicases
have been suggested, but not proven, to participate in small regulatory RNA events.
We have looked at a mutant lacking one DExH-box RNA helicase in L. monocytogenes.
This DExH-box RNA helicase deletion strain displayed an inability to grow
at 4 °C and had an impaired growth at temperatures below 37 °C. Motility was also
affected in the deletion strain implying that it cannot spread in food sources as
well as the wild-type strain. All phenotypes were rescued in a complemented
strain. Our results indicate that RNA helicases is of high importance to the bacterium
for growth at lower temperatures, and provides a link between RNA helicases
and motility.
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AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
A Listeria monocytogenes strain is still virulent despite non-functional
major virulence genes: Optical Mapping shows a potential mechanism
Roche, S. M. 1, Grépinet, O. 1, Corde, Y. 1, Teixeira, A. P. 1, Kerouanton, A. 2,
Témoin, S. 1, Mereghetti, L. 3, Brisabois, A. 2 and Velge, P. 1
1. INRA, UR 1282 Infectiologie Animale et Santé Publique, F-37380 Nouzilly and IFR 136, Agents
transmissibles et Infectiologie, France
2. AFSSA LERQAP, Unité Caractérisation et Epidémiologie Bactérienne, F-94706 Maisons-Alfort,
France
3. Université François Rabelais de Tours, EA3854 “Bactéries et risque materno-foetal”, Tours, France
and CHRU, Tours, France
L. monocytogenes is a pathogenic species, but some strains exhibit low virulence. In
previous studies, 24 naturally occurring low-virulence L. monocytogenes strains
were identified using a method combining a plaque-forming (PF) assay with subcutaneous
(SC) injection into the left hind footpad of mice. Based on their phenotypic
characteristics, these low-virulence strains have been assigned by cluster
analysis to one of four groups. The 11 strains belonging to Group I exhibit a mutated
PrfA whereas five out of the six strains belonging to Group III have causal
mutations in plcA, inlA and inlB genes. New strains exhibiting the same PFGE specific
profile than the low-virulence Group III strains have been identified and characterized.
All were low-virulence strains exhibiting the same mutations characteristic
of the Group III strains, beside the A23 strain which has been
characterized as a virulent strain in the mouse model. Interestingly, this strain
exhibited the same mutations than the low-virulence Group III strains in the inlA,
inlB, plcA genes and another one in the mpl gene. This led to expression of inactive
InlB, PI-PLC and PC-PLC proteins and to a lack of InlA expression which are
known to be important for virulence. Despite these mutations in these major virulence
genes, the A23 L. monocytogenes strain was still virulent in vitro and in vivo.
Analysis by Optical Mapping (Phylogene-France, OpGen-USA) of the A23 and two
Group III strains in comparison with the EGDe genome showed specific fragments
inserted in the genome of the Group III strains. Identification of the genes modified
by the insertion could explain the virulence of the A23 strain compared to the
Group III strains. Phylogenetic analysis of these strains could be fruitful to understand
the evolution of virulence trait as well as plasticity of virulence genes.
Protein expression of lineage I, II, and III Listeria monocytogenes
strains in murine macrophages
Donaldson, J. R., Nanduri, B., Pittman, J. R., Burgess, S. C. and Lawrence, M. L.
Mississippi State University, USA
In the current study, we compared the ability of Listeria monocytogenes strains from
genetic lineages I, II, and III (serovar 1/2a strain EGD, serovar 4b strain F2365, and
serovar 4a strain HCC23) to proliferate in the murine macrophage cell line J774.1.
We found that the lineage III strain HCC23 was able to initiate an infection but
could not establish prolonged infection within the macrophages. By contrast, strains
EGD and F2365 proliferated within macrophages for at least 7 hr. We further characterized
this interesting phenotypic difference by determining the protein expression
profiles of these strains at 0 hr, 3 hr, and 5 hr post-infection using two dimensional
liquid chromatography coupled with electrospray ionization tandem mass
spectrometry. We found that metabolic and cell wall associated proteins were expressed
by all three strains at 3 hr post-infection. However, increased expression of
stress response and DNA repair proteins was detected in the lineage I and II strains
at 5 hr post-infection. These proteins were not significantly increased in the lineage
III strain at this time point. By comparing the protein expression patterns of these
three L. monocytogenes strains during intracellular growth in macrophages, we were
able to detect biological differences that may determine the ability of L. monocytogenes
to survive and persist in macrophages.

AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
Prevalence of L. monocytogenes in bovine mastitic milk samples:
Possible source of food borne infection
Kalorey, D. R. 1, Warke, S. 1, Kurkure, N. V. 1 and Barbuddhe, S. B. 2
1. Nagpur Veterinary College, India
2. ICAR Research Complex for Goa, India
Listeria monocytogenes is a food borne pathogen responsible for listeriosis, disease
characterized by meningitis, encephalitis, reproductive disorders and septicemia
in human beings. One of the major source of Listeria is milk. A study was
conducted to know the prevalence of Listeria monocytogenes in mastitic milk from
central India. Quarter milk samples (n = 1221) were collected from dairy cows and
screened for sub clinical mastitis by California mastitis test (CMT). CMT positive
samples were examined for the presence of L. monocytogenes following two step
enrichment and plating on selective agar. Confirmation of isolates was based on
biochemical tests, haemolysis on blood agar and CAMP test. The L. monocytogenes
confirmed isolates were subjected to PCR assay for detection of virulence marker
genes (hlyA, actA, iap and prf A). A total of 71 (5.81 %) milk samples harbored L.
monocytogenes. The hlyA gene was detected in all the isolates. The hlyA and prfA
genes were detected in 33 strains. The hlyA and actA were in seven strains. Eleven
strains harbored hlyA, actA and iap genes. All isolated strains were subjected to
serotyping by PCR and revealed all to be of 4b, 4d and 4e serogroup. Excretion of L.
monocytogenes in milk may contribute to food borne infection in human. Detection
of 4b serogroup is of highly significance owing to its association with food borne
listeriosis outbreaks.
Agr-dependent peptide sensing in L. monocytogenes – Effects on biofilm
formation, virulence and global gene expression
Waidmann, M. S. 1*, Monk, I. R. 2, Auchter, M. 1, Preising, N. P. 1, Hill, C. 3 and Riedel, C. U. 1
1. Institute of Microbiology and Biotechnology, University of Ulm, Germany
2. Trinity College Dublin, Ireland
3. University College Cork, Ireland
Autoinducing peptides are signalling molecules used in population-dependent gene
regulation of Gram-positive microorganisms. The best studied example of bacterial
peptide sensing is the agr system of staphylococci. An operon with high structural
and sequence similarity to the staphylococcal arg system was recently identified in
the genome of L. monocytogenes EGDe. The listerial agr system is composed of a four
gene operon with agrB involved in the proteolytic processing/export of the gene
product of agrD, the posttranscriptionally modified signalling peptide, and
agrA/agrC encoding a typical two-component system with histidine kinase (AgrC)
and response regulator (AgrA). Here, we present our recent advances in identifying
and characterizing the native agr peptide of L. monocytogenens from spent cell culture
supernatant and the effects of agr peptide sensing on biofilm formation, virulence
and global gene expression. Furthermore, the mechanisms of agr-dependent
gene regulation in L. monocytogenes were investigated. A ∆argD mutant showed a
significant defect in biofilm formation. Invasion of EGDe ∆argD into Caco-2 and
Hep-2 cells was significantly reduced compared to EGDe wt. Moreover, the ∆argD
mutant showed an unusual subcellular distribution after primary invasion. In line
with these observations promoter activities of important virulence genes were reduced
in the ∆argD mutant. Using bioluminescence in vivo imaging, a significant attenuation
of EGDe ∆argD in a murine model of listeriosis could be shown. Moreover,
microarray analysis revealed that expression of a large number of genes
belonging to all functional categories was affected in EGDe ∆argD both in exponential
and stationary growth phase indicating a global impact of agr-dependent peptide
sensing on all aspects of physiology in L. monocytogenes.
* Participation Supported by IUFoST.
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AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
agrD-deletion affects InlA- and InlB-regulation via
a temperature-dependent and -independent mechanism
Waidmann, M. S. 1*, Monk, I. R. 2 and Riedel, C. U. 1
1. Institute of Microbiology and Biotechnology, University of Ulm, Germany
2. University College Cork, Ireland
Quorum sensing via the secretion of autoinducing peptides is a commonly used
mechanism for gene regulation among Gram-positive bacteria. A well known example
is the accessory gene regulator (agr) in Staphylococcus aureus, which is
known to affect virulence. Following the sequencing of Listeria monocytogenes
EGDe genome and the identification of a homologous gene locus, suggestions of a
regulatory role in virulence arose. The first step of listerial pathogenic lifecycle is
the entry into a host cell, a process mediated by members of the Internalin family.
Therefore, Internalin A and B are described as the main players for uptake by epithelial
and endothelial cells. To testify the role of agr in regulation of these virulence
factors, we evaluated the invasive capability of an agrD deletion mutant,
which lacks the putative autoinducing peptide, into Caco-2 and HEp-2 cells. These
assays showed that the Internalin A-dependent invasion of the mutant was significantly
decreased in comparison to the wild type. The temperature chosen for
growing pre-cultures did not influence this defect. By contrast, the effect of the
agrD deletion for Internalin B-mediated invasion into HEp-2 cells was only impaired
in pre-cultures grown at 37 °C. Furthermore, the total numbers of invaded
bacteria were much higher as compared to 30 °C. These results indicate a temperature-independent
role of agr in the regulation of Internalin A and a temperaturedependent
mechanism in case of Internalin B. This influence could occur via affecting
either the expression itself or the activation of the identified regulators
PrfA and σ B. According to these results agr seems to be an additional player for listerial
virulence.
* Participation Supported by IUFoST.
Bovine cranial nerve Schwann cells express E-cadherin, a candidate
key-player in the brainstem invasion of Listeria monocytogenes in cattle
Madarame, H. 1, Seuberlich, T. 2, Vandevelde, M. 2, Zurbriggen, A. 2, and Oevermann, A. 2
1. Veterinary Teaching Hospital, Azabu University, Japan
2. Neurocenter, Vetsuisse Faculty Bern, Switzerland
The interaction of internalin A, a major surface ligand of Listeria monocytogenes
(LM), and its host cell-receptor E-cadherin is required for the entry of LM into
cells and for the crossing of the intestinal and placental barrier during infection. In
ruminants, listeriosis occurs most commonly as rhombencephalitis, specifically
targeting the brainstem, and it is believed that LM enters the brain via cranial
nerves. However, the host cell receptors involved in brain invasion are not known.
The aim of this study was to investigate the expression of E-cadherin in cattle
brain, trigeminal nerve (TN) and its ganglion (TG), and thus its putative role in
brain invasion. To this end, brains, TN and TG of cattle were examined by immunohistochemistry
(IHC, n = 6) and Western blot (n = 2). In the brain, strong
membranous E-cadherin expression was observed in choroid plexus epithelial
cells by IHC, whilst no other brain structures were labeled. TN and TG exhibited
strong E-cadherin expression in Schwann cells and satellite cells (which are specialized
Schwann cells), respectively. The labeling was particularly evident at the
intercellular boundary of satellite cells and at the Schmidt-Lantermann incisures.
Confirming the IHC results, E-cadherin specific bands of molecular masses of approximately
37 kDa and 130 kD were evident in the WB of the TG, but were absent
in the WB of the brainstem. These results indicate that E-cadherin expressing
Schwann cells of cranial nerves might be the initial port of entry for LM in cattle
rhombencephalitis. From there, LM might gain access to the brain by cell-to-cell
spread via axons. However, this hypothesis needs to be confirmed by complementary
in vitro investigations of the molecular host-pathogen interactions.

AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
Pathogenic potential of Listeria monocytogenes isolates
from New Zealand seafood premises: implications for control
Durante Cruz, C. and Fletcher, G.
The New Zealand Institute for Plant and Food Research
Listeria monocytogenes is likely to persist in food premises, leading to contamination
of products processed therein. It is therefore important to assess the virulence potential
of these isolates in order to evaluate the risk they can pose to consumers’
heath. In this work, we assessed the invasion capacity of L. monocytogenes isolates
obtained from New Zealand (NZ) seafood premises and correlated this with their
persistence and genetic profile. From 120 isolates obtained from four seafood premises,
30 (28 from environmental and 2 from product samples) belonging to different
serotypes and pulsotypes were compared in this study. These were compared with
five human isolates obtained from the NZ Reference Culture Collection, two isolates
(human and food) from a NZ seafood-related outbreak of listeriosis and one
meat isolate shown to be resistant to high pressure conditions. ScottA was used as a
reference strain and assigned an invasion index of 100 %. In vitro studies were conducted
using Caco-2 cells (100:1 Listeria:Caco-2) followed by 40 min incubation at
37 °C. Three independent experiments were performed on each isolate in duplicate.
In Greenshell mussel premises, isolates from Plant I showed higher invasiveness
than those from Plant II. All isolates were serotype 1/2a, except for one 1/2b from
Plant I. The persistent and predominant pulsotype from Plant II had a low invasion
index. Serotype 1/2b and 4b isolates from the two fish premises showed higher invasiveness
than the serotype 1/2a isolates from mussel plants. Product (serotypes
1/2a and 1/2b) and one human (serotype 1/2a) isolates showed higher invasion indices
than environmental ones. The seafood outbreak strains had an intermediate
invasion index. This study improves our understanding of L. monocytogenes present
in NZ seafood premises and highlights the need to improve control. Although
some environmental factory isolates were potentially pathogenic, persistent strains
were generally less invasive in Caco-2 cells.
Copper homeostasis and virulence in Listeria monocytogenes
David, C. 1, Schuler, S. 1, Glenn, S. 2, Jen, C. 1, Andrew, P. 2 and Roberts, I. S. 1
1. University of Manchester, UK
2. University of Leicester, UK
Copper is essential for many bacteria being an important prosthetic group for certain
enzymes. However at high levels copper is toxic in part due to its ability to
redox cycle and catalyse the formation of oxygen derived free radicals. As such
bacteria need to be able to maintain copper homeostasis. In L. monocytogenes the
problem of copper homeostasis is particularly acute. It inhabits a range of environments
including soil, effluents and food where it will be exposed to fluctuating
copper levels. The use of copper as an anti-microbial in animal feeds, as a disinfectant
in factory-based farming and as a biocide in fruit production means L.
monocytogenes will have to combat potentially toxic levels of copper during food
production and processing. Adapting to fluctuations in the level of copper is important
during infections. L. monocytogenes replicates in a number of host tissues
that will offer different challenges with regard to copper availability. Early in infection
L. monocytogenes grows in the gall bladder, where there are high levels of
copper, whereas in the liver and spleen it replicates in an environment in which
there may be little free copper. Adapting to these different niches within the host
requires effective copper homeostasis. In this paper we describe and analyse the
operon that encodes for the only P1 type-ATPase copper exporter in L. monocytogenes
and describe the molecular basis by which transcription of this operon is
regulated by environmental copper and identify the role played by this operon in
infection of L. monocytogenes.
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AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
Pattern of cytokine production during murine listeriosis
Dussurget, O.
Institut Pasteur, France
Listeria monocytogenes causes listeriosis, an infection whose manifestations extend
from gastroenteritis to septicemia, meningitis, encephalitis, abortions and
perinatal infections. Cytokines are important mediators of host defense against
pathogenic microorganisms. They control innate immune responses and contribute
to shape adaptive immune responses. Several studies have previously analyzed
the production of one or several cytokines in response to Listeria monocytogenes
in vitro and also in vivo. In this work, we used a murine model of primary
listeriosis to systematically follow in the same animal the protein levels of 20 cytokines
in the blood and in the liver and spleen, two important organs in which L.
monocytogenes replicate. Infection was shown to trigger the secretion of the major
activator of macrophage antimicrobial activity, interferon (IFN) g, in blood, liver
and spleen 24 h after intravenous inoculation. The early response to infection was
also characterized by a significant increase in proinflammatory cytokines tumor
necrosis factor (TNF) a, interleukin (IL) 1a, IL1b and IL6, chemokines KC, IP10,
MCP1, MIP1a and MIG, and growth factors GM-CSF and V-EGF. Secretion of cytokines
important for the T cell response, e.g. IL2, and more specifically for T
helper 1 (Th1) cell response, IL12 and IL10, was induced after infection. Surprisingly,
the level of IL4 and IL13, two cytokines involved in Th2 cell response, also
increased upon infection. The level of most cytokines increased in correlation with
the number of bacteria in each organ during the first three days of listeriosis. In
contrast, a strain of L. monocytogenes which does not produce the major virulence
factor listeriolysin O (LLO), and is cleared very early from infected mice, failed to
stimulate cytokine production. Together, this first multiplex analysis of cytokine
production in vivo represents an important basis to identify virulence determinants
involved in the modulation of host immune response.
Analysis of the post-translocation chaperone PrsA2 and its unique role
in facilitating Listeria monocytogenes pathogenesis
Alonzo, F. and Freitag, N.
University Of Illinois at Chicago, USA
Listeria monocytogenes is a Gram-positive bacterial pathogen whose ability to
cause disease is dependent upon the coordinated expression and activity of secreted
products that promote virulence. We have shown that the post-translocation
chaperone PrsA2 is critical for L. monocytogenes pathogenesis and that prsA2
mutants exhibit a substantial cell-to-cell spread defect in tissue culture. We therefore
hypothesized that a loss of PrsA2 results in perturbation and/or altered activity
of factors responsible for intracellular survival. PrsA2 was found to promote
the activity and stability of two secreted virulence factors: listeriolysin O (LLO)
and the broad range phospholipase PC-PLC. Culture supernatants from strains
containing deletions of prsA2 exhibited reduced hemolytic activity as measured
by the lysis of red blood cells in vitro, and this reduced activity appeared to be the
result of altered stability and increased proteolytic cleavage of LLO. prsA2 mutants
were also defective for processing of PC-PLC from its pro-form to its enzymatically
active mature form, resulting in decreased phospholipase activity in vitro
as well as within infected host cells. PrsA2’s role in bacterial virulence was found to
be unique from the related L. monocytogenes chaperone PrsA1, a protein that
shares a high degree of sequence similarity with PrsA2 but makes no discernable
contribution to pathogenesis. Proteomic analysis of L. monocytogenes secreted
proteins suggests that PrsA2 is required for the correct localization of additional
bacterial gene products. Taken together, these data indicate that PrsA2 has been
functionally adapted to promote the secretion and activity of multiple L. monocytogenes
factors that play important roles in bacterial virulence.

AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
CtaP is a multifunctional cysteine-transport associated protein
required for Listeria monocytogenes pathogenesis
Xayarath, B.
University of Illinois at Chicago, USA
The bacterial pathogen Listeria monocytogenes survives under a myriad of conditions
in the outside environment and also within the human host where infections
can result in severe disease. Bacterial life within the host requires the expression
of genes with roles in nutrient acquisition as well as the biosynthesis of bacterial
products required to support intracellular growth. A gene product identified as
the substrate-binding component of a novel oligopeptide transport system (encoded
by lmo0135) was recently shown to be required for L. monocytogenes virulence.
We report here that the gene product encoded by lmo0135 makes multiple
contributions to L. monocytogenes growth and survival in environments both inside
and outside of host cells. The lmo0135 gene product was required for bacterial
growth in the presence of low concentrations of cysteine in vitro, suggesting
lmo0135 and its associated transport system function in high affinity cysteine
transport. We have therefore designated the lmo0135 gene products as CtaP for
cysteine-transport associated protein. Interestingly, CtaP was not required for
bacterial replication within the host cytosol, but was required for bacterial adhesion
implicating a role for this protein in bacterial attachment to host cells. CtaP
appears to function directly as an adhesin as latex beads coupled to CtaP were
found to specifically bind epithelial cell monolayers in tissue culture assays. In
addition, loss of CtaP increased membrane permeability and acid sensitivity, resulted
in altered bacterial surface hydrophobicity, and severely attenuated virulence
following both intragastric and intravenous inoculation of mice. Taken together,
the data presented indicates that CtaP is part of a unique ABC transport
system that contributes to multiple facets of L. monocytogenes physiology, growth,
and survival both inside and outside of animal cells.
Enzymatic activity of the metalloprotease of Listeria is regulated by pH
Forster, B. M.*, Pavinski Bitar, A., Slepkov, E. R. and Marquis, H.
Cornell University, USA
The broad-range phospholipase C of Listeria monocytogenes, PC-PLC, contributes
to escape from vacuoles. During infection, PC-PLC accumulates at the membrane
cell wall interface until a decrease in vacuolar pH triggers its proteolytic maturation
and release. PC-PLC maturation is mediated by the metalloprotease of Listeria
(Mpl), which is produced as a zymogen and matures via intramolecular autocatalysis.
We tested the hypothesis that Mpl activity is regulated by pH. Radiolabeled
infected cells were perfused to manipulate host cell cytosolic pH, and secreted Mpl
was immunoprecipitated. Mature Mpl was detected in samples treated at pH 6.5,
but not at pH 7.3. To distinguish whether pH regulates autocatalysis or cell wall
translocation of bacterium-associated Mpl, we inserted a Flag tag at the N-terminus
of the pro or catalytic domain of Mpl, and used an antibody that recognizes only
N-terminal Flag to detect Mpl. Results from fluorescence microscopy indicated
that the zymogen is bacterium-associated at physiological pH, but not following
acidification of the host cell cytosol. Mature Mpl was not detected bacterium-associated
at either pH. Lastly, using purified mature Mpl and the proform of PC-
PLC, we determined that processing of the PC-PLC propeptide by mature Mpl occurs
only at acidic pH. Together, these results indicated that Mpl enzymatic activity
is regulated by pH, whether it undergoes autocatalysis or mediates the maturation
of PC-PLC. Future studies will aim at assessing the possibility that specific amino
acid residues act as pH sensors in the regulation of Mpl autocatalysis.
* Participation Supported by IUFoST.
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Identification of propeptide residues regulating the
compartmentalization, maturation, and activity of the broad-range
phospholipase C of Listeria monocytogenes
Slepkov, E. R., Pavinski Bitar, A. and Marquis, H.
Cornell University, USA
The broad-range phospholipase C of Listeria monocytogenes, PC-PLC, is made as
an inactive proenzyme whose activation is regulated by pH and by the metalloprotease
of Listeria (Mpl). The propeptide of PC-PLC is comprised of 24 amino
acid residues (C28-S51) and regulates PC-PLC compartmentalization and activity.
In this study, we generated a series of propeptide mutants to determine the minimal
requirement to prevent PC-PLC activity, and to identify amino acid residues
regulating compartmentalization and maturation. To determine how many
residues were required to prevent PC-PLC activity, nested deletions were generated
in the PC-PLC propeptide of an Mpl-minus strain and phospholipase activity
was assessed on egg yolk plates. We found that a single amino acid residue at position
P1 (S51) of the cleavage site is sufficient to prevent activity, which is consistent
with P1’ (W52) being located within the active site pocket. The requirements to
retain the proform of PC-PLC bacterium-associated were assessed by immunoprecipitating
secreted PC-PLC from radiolabeled infected cells maintained at
physiological pH, whereas the influence of the propeptide on the ability of Mpl to
mediate PC-PLC maturation was assessed by immunoprecipitating secreted PC-
PLC from radiolabeled infected cells perfused with buffer at acidic pH. We observed
that the triple mutant E31A Y32A L33A is translocated across the cell wall
more effectively than wild-type at physiological pH, and that Y32A and L33A single
point mutants are less effectively processed by Mpl at acidic pH. These results
indicated that the N-terminus of the propeptide regulates PC-PLC compartmentalization
and the ability of Mpl to process PC-PLC. Considering that Y32 and L33
are located 19 and 18 residues upstream of the propeptide cleavage site, we suggest
that these two residues initiate the interaction with mature Mpl leading to processing
of the propeptide at S51.
A mouse model of fetoplacental Listeria monocytogenes infection
and abortion
Poulsen, K. P., Faith, N., Laura Knoll, L. and Czuprynski, C.
School of Veterinary Medicine, University of Wisconsin Madison, USA
Foodborne outbreaks of Listeria monocytogenes, particularly with serotype 4b,
continueto occur. Pregnant women are over represented in listeriosis outbreaks
(17-fold increasein incidence of disease). Infection of the fetus and placenta results
in abortion, stillbirth,or premature parturition. The latter carries with it a
high risk for neonatal sepsis andmeningitis. Alterations in cellular and molecular
components of the immune systemoccur during pregnancy to prevent rejection
of the maternal allograft (i.e. fetus). How these pregnancy associated changes affect
vertical transmission of L. monocytogenes to the fetus, is poorly understood.
We have developed a mouse model for infection with aserotype 4b strain of L.
monocytogenes isolated from a foodborne listeriosis outbreak thatresulted in abortion
and fetal death. Intragastric infection of pregnant mice resulted insevere infection
of the maternal and fetal tissues, in both C57BL/6J and A/J mousestrains.
Use of a luciferase tagged L. monocytogenes strain and bioluminescent technology
allowed us to visualize infection of maternal and fetal tissues and shedding of L.
monocytogenes cells in feces and vaginal secretions of aborting mice. These data
support the use of pregnant mice as a model for maternal and fetal L. monocytogenes
infection. This model will prove useful in future investigations of the maternal
and fetal immune response to listeriosis during pregnancy.

AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
Listeria infection of the insect model system Galleria mellonella
Joyce, S. A. and Gahan, C. G.
APC and department of Microbiology UCC, Ireland
With the exception of mice, alternative model systems are constrained by an upper
temperature limit for their survival. This temperature varies from 25 °C for Acanthamoeba
and Danio rerio to 28 °C for C. elegans and for Drosophila species. This
limitation questions the relevance of these models for use with human pathogens
where virulence gene expression is temperature dependent. Galleria mellonella,
the Greater Wax Moth Larvae, is well documented as an infection model amenable
to elevated temperature of 37 °C. Here, we examine and establish Galleria mellonella
as a relevant model for infection by Listeria species. Mutants attenuated for
virulence in mice are similarly attenuated in this model system. We demonstrate
that haemolysin production is the dominant factor in the pathogenic process of L.
monocytogenes to G. mellonella. Real time visualization of infection reveals that the
same sub-sets of virulence genes required for L. monocytogenes infection of both
humans and mice are expressed (differentially) and required for insect infections.
L. monocytogenes replication occurs in G. mellonella. On examining the host response
we find that L. monocytogenes is mainly internalized within one hour of infection.
In the presence of hemolysin, hemocyte viability decreased eight- fold and
in the absence of PrfA a four-fold increase in viability was evident relative to WT
strain. Bacterial load in hemocytes was significantly increased on infection with a
mutant in hemolysin (DhlyA) production and a mutant in stress factor production
(DsigB) compared to wt infection. We found that the Phenyloxidase (PO) system
mounts a response to the presence of Listeria species within 4 hour of insect infection
and that infection by L. monocytogenes results in the production of anti microbial
peptides (AMPs). Taken together, we propose that G. mellonella is a relevant
model for infection by L. monocytogenes at 37 °C.
Construction of a murinised Listeria monocytogenes H7858 (4b) strain
for improved murine infection
Cummins, J. and Gahan, C.
Science Foundation Ireland
Listeria monocytogenes is Gram-positive food borne pathogen that is capable of
causing listerosis culminating in various diseases in humans such as gastroenteritis,
meningitis, encephalitis and spontaneous abortions. L. monocytogenes is capable
of internalisation into non-phagocytic cells and crossing the intestinal, the
placental and the blood-brain barriers. Internalisation into non-professional
phagocytic cells is mediated in particular by the surface protein, InlA. InlA promotes
listerial uptake into enterocytes by targeting the N-terminal domain of the
human E-cadherin. However, this interaction is species specific with its interaction
completely impaired in the rat and mouse models due to the absence of a proline
at position 16 of the N-terminal E-cadherin repeat. A landmark result was the
development of a transgenic mouse line expressing human E-cadherin by intestinal
enterocytes. However, an alternative approach to overcome the lack of appropriate
animal models has been the creation of a murinised strain of L. monocytogenes
EGDe (Wollert et al., 2007 Cell. 129(5):891-902). By the substitution of
two amino acids within the InlA protein we have created a murnised strain in the
H7858 4b background. Our results have demonstrated that this mutant has an increased
ability to infect mice by the oral route and does not increase invasion
within human cells. The benefits of this approach will be discussed.
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AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
The role of a phosphoinositide phosphatase in the intracellular survival
of Listeria monocytogenes
Wang, J., Corbett, D. and Roberts, I. S.
Faculty of Life Sciences, University of Manchester, UK
L. monocytogenes is capable of invading and growing in a number of host cells. Following
uptake it escapes from the phagosome and grows in the cytosol recruiting
actin filaments to move and spread from cell to cell. Once inside the cell it subverts
the host cell physiology to promote its own growth and survival. Phosphoinositides
are phospholipids present in host cell membranes that play key regulatory
roles in orchestrating cell physiology. They control a broad range of processes
including organelle identity, signal transduction and cell migration. The basis of
this control is their relative state of phosphorylation that is controlled through
the action of host kinases and phosphatases. The central role played by phosphoinositides
inside cells make them a prime target for subversion by intracellular
pathogens wishing to hijack the cell’s physiology. Recently we discovered that L.
monocytogenes expresses a phosphoinositide phosphatase, capable of removing
phosphate groups from a number of important phosphoinositides. Importantly
we demonstrated that this enzyme was essential for the growth of L. monocytogenes
inside infected cells and probably is important in escaping the phagosome.
This is the first identification of a phosphoinositide phosphatase enzyme playing
a key role in the intracellular survival of L. monocytogenes.
Virulence gene expression in Listeria monocytogenes strains isolated
from different sources
Alessandria, V.
University of Turin, Italy
Listeria monocytogenes is an important food-borne pathogen that has the capacity
to cause severe infections. Ubiquitous microorganism, it is commonly isolated
from foods of animal origin, mainly meat and milk products. However, due to its
capacity to develop at refrigeration temperatures, human listeriosis outbreaks are
most often associated with ready-to-eat food products that are consumed without
prior cooking. The incidence of the disease depends on different factors including
the infective dose and the immunity conditions of the host. The present
work focuses on the expression analysis of four virulence genes (sigB, plcA, hly
and iap) in 11 different strains of L. monocytogenes and, more in detail, 3 collection
strains (EGDe, NCTC10527, SCOTT A), 7 isolated from food matrices (4 of which
isolated from meat products and three from diary products) and 1 isolated from
humans. In the first step, the expression analysis was performed in vitro, culturing
each strain in BHI (Brain Heart Infusion) medium, in order to identify possible
differences in the expression level for the different strains. The combined use of
reverse transcription and quantitative PCR (qRT-PCR) was used to evaluate gene
expression. As a second step, we wanted to evaluate the trend of expression of the
genes in food. Analyses were performed inoculating L. monocytogenes in milk at
different conditions and in particular at two temperatures (4 and 12 °C) for different
times (24 and 48 hours). Significant expression differences emerged for the
different genes, without showing any significant association between the expression
of the genes and the origin of the different strains.

AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
Targeted Signature-tagged mutagenesis for phenotype screening
of Listeria monocytogenes mutants
Henriques, A., Carvalho, F. and Cabanes, D.
IBMC, Portugal
Listeria monocytogenes is a foodborne facultative intracellular human pathogen
possessing a wide range of virulence factors tailored for pathogenesis. Common
strategies used to identify new virulence factors are based on mutagenesis and in
vivo phenotype analysis, which requires testing of generated mutants versus wild
type strain in separate mice cohorts. To streamline this analysis, a targeted signature-tagged
mutagenesis (T-STM) method was developed. Based on the concept of
STM, where random mutants identifiable by specific tagging are negatively selected,
we developed a method that enables specific gene targeting in L. monocytogenes
genome for the creation of insertional mutants and their simultaneous
phenotypic analysis in mice. Tagged “wild type” and mutants for prfA and two unknown
LPXTG genes were constructed by inserting differentially tagged suicide
vectors. T-STM uses defined tags that allow the discrimination between taggedstrains
in a pool by PCR. The four tagged-strains were pooled in equivalent
amounts into one single inoculum (Input) used for oral and intravenous infection
of mice. Three days after infection, intestine, spleen, and liver of mice were collected,
homogenised and plated on rich medium. Resultant bacterial growth (Output)
was used as template for PCR screening and evaluation of the relative proportion
of each tagged-strain. Mutants were considered less virulent when
undetectable (or less detectable) by PCR in the output. This technique allowed us
to clearly distinguish the severely attenuated prfA mutant, validating the method.
PCR products for the two LPXTG protein-encoding mutants appeared in equal
proportion to the wild type strain in the output, suggesting that these proteins are
not involved in Listeria virulence in this infectious model. This study shows the
feasibility and advantage of the developed T-STM method for simultaneous virulence
analysis of pools of Listeria tagged mutants. This approach should be now
used for larger-scale phenotypic analysis of specific L. monocytogenes mutants.
Listeria monocytogenes cellular infection triggers tyrosinephosphorylation
of Myosin IIA, a new protein involved in invasion
Almeida, M. T., Cabanes, D. and Sousa, S.
IBMC, Portugal
Listeria monocytogenes is a human food borne pathogen that may lead, in particular
in immunocompromised individuals, to a severe disease characterized by septicemias,
meningitis, meningo-encephalitis and abortions. The study of the cell
biology of the Listeria infectious process provided insights in the way bacteria manipulate
the host and revealed unsuspected functions of cellular proteins. To cause
infection pathogens interfere with crucial host intracellular pathways, and different
pathogens often hijack the same signaling pathways. In particular, host phosphorylation
cascades are preferential targets of infecting bacteria. In this study,
using L. monocytogenes as a pathogen model, we showed that eukaryotic cells present
a variable protein phosphorylation pattern upon infection. We addressed in
particular the tyrosine-phosphorylated protein profile triggered by Listeria infection
and identified the motor protein, Myosin IIA (MyoIIA), as differentially tyrosine-phosphorylated
in response to Listeria uptake. We demonstrated that MyoIIA
is not only tyrosine-phosphorylated over the time of infection, but is also
recruited with actin at the bacteria entry site. In addition, we were able to show
that the inhibition of MyoIIA activity affected Listeria entry into non-phagocytic
cells. The reduction of MyoIIA expression using RNAi techniques resulted in an
increased Listeria uptake. Together these data point to the role of a novel myosin
class in the internalization of Listeria, correlating for the first time, myosin posttranslational
modifications and Listeria infection.
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AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
Invasion profile of Listeria monocytogenes strains involved
in invasive and gastroenteritis listeriosis outbreaks
Laksanalamai, P., Sahu, S. and Datta, A.
Food and Drug Administration, USA
Listeria monocytogenes (Lm), the causative agent of foodborne human listeriosis, is
a serious public health concern. The disease is characterized by meningitis, septicemia,
abortion and death in immuno-compromised population. Growing numbers
of evidence have revealed that Lm can also cause self-limited febrile gastroenteritis
in healthy individuals. To understand the mechanisms underlying two
different disease outcomes several efforts have been made to differentiate gastroenteritis
and invasive strains of Lm. Since the difference between these two
types of outbreaks is Listerial ability to invade, in this study we have examined
the expression of two internalin genes, inlA and inlB which encode virulence proteins
required for the internalization process. The inlA and inlB gene expression
profiles were determined by quantitative RT-PCR from several strains representing
two groups of Lm that cause either invasive listeriosis or febrile gastroenteritis.
Analysis of the inlA and inlB gene expression from food and patient isolates
showed similar levels of expression from isolates originated from the same outbreaks.
Surprisingly, the inlA and inlB gene expression from Lm isolated from invasive
listeriosis outbreaks appears to have lower expression that those that
caused gastroenteritis listeriosis. In contrast the expression of iap, a gene involved
in host cell invasion, was higher in invasive strains. To understand the correlation
between the inlA and inlB gene expression and invasion of Lm, we also investigated
the efficiency of invasion of these strains by in vitro invasion assay using eukaryotic
cell lines, Caco2 and HepG2. The results appeared to correlate with the
inlA and inlB expression in that the invasion efficiency is higher in the gastroenteritis
strains. Global gene expression profiles of these strains are currently being
investigated using a DNA microarray.
Molecular characterization of the Vip-Gp96 interaction
Martins, M., Cabanes, D. and Sousa, S.
IBMC, Portugal
The study of mechanisms exploited by Listeria monocytogenes to cause infection
provided new insights in the way bacteria manipulate the host cell machinery. To
subvert the host cell signalling pathways, Listeria uses a complex set of surface
proteins called virulence factors that act in concert to allow bacterial infection
and evasion from the host immune system. Recently, we reported that Vip, a L.
monocytogenes virulence factor interacts with Gp96, an endoplasmic reticulum
(ER) resident chaperone that in cell stress conditions is targeted to cell surface,
promoting host cell invasion. The purpose of this study was the identification of
the Gp96 domain involved in the interaction with Vip. Using protein-protein interaction
and invasion assays, we demonstrated that the region 22-411 amino acids
of Gp96 (N-terminal) seems to be exposed outside of the cell thus available for
Vip binding. This interaction is required for Listeria uptake into host cells. Although
this entire region participates in the interaction, the domain comprising
the 22-192 amino acids seemed to be the one crucial for the interaction. The existence
of cell signalling events downstream Vip-Gp96 interaction was also investigated.
Signal transducer and activator of transcription 3 (Stat3) activation during
L. monocytogenes invasion was addressed, suggesting that Stat3 was not activated
in response to Listeria entry. In addition, we demonstrated that Vip seems to have
no role in Listeria phagocytosis by bone marrow-derived macrophages (BMMØ)
and in bacterial survival within them.

AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
Investigation of the molecular mechanisms by which
Listeria monocytogenes grows in the mammalian gall bladder
Dowd, G., Joyce, S., Casey, P. G., Hill, C. and Gahan, C. G.
University College Cork, Ireland
Listeria monocytogenes is a foodborne pathogen that is a significant cause of mortalities
due to consumption of contaminated foods. Recent work has demonstrated
that the pathogen grows in the mammalian gall bladder and that gall bladder growth
forms a significant phase of the infectious process. However, relatively little is
known about the mechanisms that underpin the growth of the pathogen in this milieu.
Here we have performed basic experiments which demonstrate that L. monocytogenes
grows well in bile from the porcine gall bladder and can utilize bile as a
source of energy and nutrients. We used bioluminescence-labeled L. monocytogenes
to allow visualization of bacterial growth kinetics in porcine gall bladders infected
ex vivo. We then performed random mutagenesis of L. monocytogenes and selected
mutants that grow well under laboratory conditions but fail to grow in bile. Sequencing
of the genes mutated in this experiment demonstrated that the pathogen
requires genetic loci that are involved in central metabolism for growth in bile, including
genes encoding proteins required for amino acid biosynthesis and biotin
metabolism. The majority of mutants that were unable to grow in bile were also
attenuated for virulence in the mouse model of infection. Overall the work is the
first to demonstrate the requirement for specific gene sets for the growth of L.
monocytogenes in the specialized environment of the mammalian gall bladder.
Role of cadmium efflux system in Listeria monocytogenes virulence
Camejo, A. and Cabanes, D.
IBMC, Portugal
Listeria monocytogenes is a human intracellular pathogen able to colonize host
tissues after ingestion of contaminated food, causing severe invasive infections.
In order to gain a better understanding of the nature of host–pathogen interactions,
we studied the L. monocytogenes genome expression during mouse infection.
We found that the shift of the Listeria genome expression during infection is
characterized by the activation of genes involved in virulence, stress, subversion of
the host immune system, and by the adaptation of the bacterial metabolism to
host conditions. Mutagenesis of genes highly induced in vivo allowed the identification
of novel L. monocytogenes virulence factors, including cadC. Cadmium resistance
in Listeria and other gram positive bacteria is an energy-dependent cadmium
efflux system, involving two proteins, CadA and CadC. CadA has been shown
to be a cadmium efflux P-type ATPase; cadC encodes a transcriptional regulator
that is a member of the ArsR metalloregulatory proteins. The strong in vivo activation
of cadC and the significant impaired virulence of the cadC mutant suggest
that this heavy metal resistance system constitutes an advantage for in vivo Listeria
survival. In addition, this system has never been previously implicated in the
virulence of other pathogens. The detailed regulation of the CadAC system role as
well as its role in cadmium resistance and in the Listeria infectious process will be
presented and discussed.
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Investigation of chitinase as a potential virulence factor
in Listeria monocytogenes
Chaudhuri, S.
University of Illinois in Chicago, USA
Listeria monocytogenes is an environmental pathogen that causes disease in humans
primarily following the consumption of contaminated food products. While
considerable attention has been given to the identification of bacterial virulence
factors specifically expressed within host cells, relatively little is known regarding
gene products that may contribute to bacterial life in the outside environment
while also potentially serving a role for virulence within the host. L. monocytogenes
has been shown to produce and secrete two chitinases, ChiA and ChiB, as
well as a chitin binding protein encoded by lmo2467. ChiA and ChiB are chitin degrading
enzymes that are presumed to facilitate L. monocytogenes life in the outside
environment where chitin is plentiful. Recently however, chitinases and chitin
binding proteins have been reported to contribute to the pathogenicity of two unrelated
environmental bacterial pathogens, Legionella pneumophila and Vibrio
cholerae. The chitinases produced by these bacteria appear to enhance bacterial
colonization of mouse lung (L. pneumophila) and intestine (V. cholerae). To determine
if chitinases or chitin binding proteins contribute to L. monocytogenes pathogenecity,
in-frame deletion mutants were constructed in chiA, chiB, and lmo2467,
and a triple gene deletion mutant was also constructed. All mutants were found to
grow similarly to wild type L. monocytogenes in BHI broth culture and within infected
tissue culture cells. However, following intravenous infection of mice, two of
the single mutants, ∆chiA and ∆chiB, as well as the triple mutant, ∆chiA ∆chiB
∆lmo2467 were found to be defective for bacterial growth in the livers and spleens
of infected animals. As mammals do not produce chitin, these results suggest that
L. monocytogenes has adapted its chitinases to exploit recognition of host substrates,
potentially carbohydrate-linked molecules, to enhance bacterial survival
within infected animals.
Sub-lethal concentrations of common disinfectants do not influence
survival and growth of Listeria monocytogenes in whole blood
Holch, A., Gaedt Kastbjerg, V. and Gram, L.
Technical University of Denmark
Listeria monocytogenes is a serious food borne bacterial pathogen that can colonize
food processing environment. Virulence gene expression is influenced by several environmental
factors e.g. temperature and oxygen-concentration. It has recently been
shown that sub-lethal concentrations of disinfectants used in the food industry affect
the expression of virulence genes in L. monocytogenes (Kastbjerg et al. 2009). The
expression is either enhanced or reduced depending on the active compound in the
disinfectant.The purpose of the study was to determine if these changes in virulence
gene expression influence the virulence potential of L. monocytogenes in a more complex
biological assay. We chose to study the ability of L. monocytogenes to grow and
survive in whole blood as several of the virulence genes are important for survival
and growth in blood. Two strains of L. monocytogenes (EGD and a maternofetal strain)
where grown to exponential phase and exposed to two different disinfectants in a
non-inhibitory and a sub-lethal concentration for one hour. The disinfectants contained
either peroxide or quaternary ammonium compound (QAC) as their active
compound. These cause enhanced or reduced virulence gene expression, respectively.
The exposed bacteria were mixed with whole blood and the survival and growth was
followed for 50 hours by plate counting. L. monocytogenes was able to grow in whole
blood after exposure to the two different disinfectants in two different concentrations.
However, pre-exposure to peroxide or QAC did not cause any difference in
growth for both strains of L. monocytogenes as compared to exposure to water (control).
In all experiments, the maternofetal strain grew slightly better than the EGD
strain. Hence, the effect of sub-lethal concentrations of disinfectants on virulence
gene expression did not affect the survival and growth in a complex biological model.

AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
Internalin LRR domain variability in Listeria monocytogenes isolated
from different hosts
Zaytseva, E. A. 1,2, Ermolaeva, S. A. 2 and Somov, G. P. 1
1. Research Institute for Epidemiology and Microbiology, RAMS Siberian Division (Vladivostok)
2. N.F. Gamaleya Research Institute for Epidemiology and Microbiology, RAMS (Moscow)
Development of certain clinic manifestations of listeriosis has been recently suggested
to depend on several factors of L. monocytogenes pathogenicity. A number
of surface and secreted proteins from internalin family, of which A and B internalin
proteins are most thoroughly investigated, are involved in the process of Listeria
invasion into eukaryotic cells. One of the specific features of this protein family
is presence of so called LLR domains involved in the direct interaction with
eukaryotic receptors. The purpose of this research is to analyze distribution of
various internalin gene alleles among L. monocytogenes isolated from different
sources. Eighty-six L. monocytogenes cultures were used belonging to serovariants
1/2a, 1/2 b, 4b, isolated from various sources in the Far East and European part of
Russia in the period from 1952 to 2005. Primers, gene restricting fragment and
coding LRR domain were selected with the help of Oligo38 application. Gene presence
in L. monocytogenes was determined with the help of PCR. DNA fragment sequencing
was performed in Genom Center (Moscow). Molecular genetics features
of Listeria were analyzed in 4 culture groups depending on isolation source: 1)
stillborn who died from listerial infection (n=21); 2) wild murine rodents (n=19); 3)
marine hydrobionts (n=19); 4) food (control, n=27). Invasion factor genes - inlA,
inlB, inlC and inlE - were found in all of L. monocytogenes isolates. Sequence of
LRR domains of genes inlA, inlB, inlC and inlE was determined for all the isolates.
L. monocytogenes isolates demonstrated specific distribution of the examined gene
alleles depending from isolation source. Among clinic isolates (group 1) low variability
was recorded for inlA and inlC (p < 0.01). L. monocytogenes isolates obtained
from wild rodent organs showed low variability with for inlB (p < 0.01). Specific
alleles of inlA, inlC and inlE were found in Listeria cultures isolated from marine
hydrobionts. Alleles of inlE were uniform in all the compared groups. In control
group all gene alleles were distributed uniformly too. The obtained results confirm
internalin role in L. monocytogenes interaction with a certain host. A microbe
with specific gene allele may be more virulent and invasive for a certain type of
host even at low infection doses.
Reduced virulence of an adenylosuccinate lyase transposon mutant
of a serotype 4b strain of Listeria monocytogenes
Faith, N. G. 1,2, Kim, J.-W. 3, Kathariou, S. 3, Sahaghian, R. 1 and Luchansky, J. B. 4
1. School of Veterinary Medicine, Univ. Wisconsin-Madison Madison, WI
2. Food Research Institute, Univ. Wisconsin-Madison Madison, WI
3. Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State Univ., Raleigh, NC
4. Eastern Regional Research Laboratory, USDA-ARS, Wyndmoor, PA
A severe outbreak of listeriosis occurred in 1998–99 as a result of contamination of
hot dogs with serotype 4b Listeria monocytogenes. We compared several characteristics
of strain H7550, implicated in the 1998–99 outbreak of listeriosis, and a plasmid-free
derivative (H7550cds) of that strain lacking the cadmium resistance plasmid
pLM80, with that of selected transposon mutant derivatives of those strains.
We assessed virulence in a mouse model of intragastric (i.g.) inoculation of anesthetized
A/J mice with approximately 10 6 CFU of the individual strains. A transposon
mutant of strain H7550 that lacks adenylosuccinate lyase activity (strain
J22F), and a non-hemolytic (LLO-) transposon mutant of strain H7550cds (J29H),
were avirulent in our mouse model. We did not recover viable cells from the spleen,
liver, blood gallbladder, or ceca of mice inoculated with either strain J22F or J29H,
whereas the respective parent strains H7550 and its cadmium sensitive derivative
H7550cds were equally virulent for mice. We observed no significant difference in
the resistance of stationary phase cells of the plasmid harboring versus plasmidfree
strains to synthetic gastric fluid at pH 4.5. All four strains formed biofilms on
plastic surfaces within 24 hr in vitro. Strain J22F was better able to form biofilms in
vitro than its parent strain H7550, whereas the LLO-negative mutant strain J29H
was not significantly different from its parent strain H7550cds in biofilm formation.
These results provide the first evidence that adenylosuccinate lyase activity is required
for virulence of L. monocytogenes in mice. They also demonstrate that LLO
is not required for biofilm formation in vitro.
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AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
Manifestations and outcome of listeriosis in adult patients
Fernández Guerrero, M L. 1, Mancebo Plaza, B. 2, Torres, R. 2, Górgolas, M. 1 and Jusdado, J. J. 2
1. Fundación Jiménez Díaz. Universidad Autónoma de Madrid, Spain
2. Hospital Severo Ochoa, Madrid, Spain
Infections caused by Listeria monocytogenes in adult patients are rarely reported
despite the increasing prevalence of these infections in western countries. Hence,
the manifestations, optimal treatment, prognosis and risk factors for mortality
are not entirely known. We herein report a review of the most relevant clinical aspects,
treatment and outcome of a large series of patients with listeriosis studied in
two hospitals in Madrid, over a period of 15 years. Patient with isolation of L. monocytogenes
from blood, CSF or other sterile fluids were included in the analysis.
Sixty-two cases were seen. The mean age was 58.5 years (from 20 to 85 years) without
differences in distribution by genders. Seventy percent of cases had comorbidities:
cirrhosis of the liver, hematologic neoplasms, immunosupressive disorders
and corticosteroid therapy were the most common. Primary bacteremia
(48 %) and meningoencephalitis (37 %) were the most common presentations. Ten
patients (16 %) presented with focal infections such as bacterial peritonitis (4),
endocarditis (3) and brain abscesses (3). Ampicillin alone or in combination with
gentamicin and cotrimoxazol were the antimicrobial treatments most frequently
used. We did not find differences in the mortality rate among patients treated with
monotherapy in comparison with those that received combined therapy. Cotrimoxazol
was successfully used in 10 out of 13 patients (77 %) treated. Twenty-three
patients (37 %) died within 30-days of diagnosis. Mortality increased according to
the severity of the underlying disease and was significantly higher in immunocompromised
patients (66 %) than in those with chronic debilitating conditions
(20 %; p < 0.001). Human listeriosis is a severe disease producing high mortality
particularly in immunocompromised patients with hematologic neoplasms and
corticosteroid therapy. Despite synergistic activity of combined ampicillin/gentamicin
therapy, the survival benefits of this combination remain theoretical. Cotrimoxazol
may be a useful alternative treatment for human listeriosis.
Model for human Listeriosis: in vivo monitoring of orally infected mice
using bioluminescent Listeria monocytogenes
Bergmann, S., Lengeling, A., Pasche, B. and Schughart, K.
Helmholtz-Centre for Infectious Research, Germany
The ubiquitous Gram-positive bacterium Listeria monocytogenes is the causative
agent of listeriosis. After ingestion of contaminated food, this pathogen is able to
cross the intestinal, blood-brain and placental barrier and leads to gastroenteritis,
meningitis and maternofetal infections which may result in abortion and spontaneous
stillbirth. We have generated a bioluminescent Listeria monocytogenes strain
which carries two amino acid substitutions in the bacterial invasion protein internalin
A (InlA S192NY369S) and enables the bacterium to bind to the murine Ecadherin
receptor with increased affinity as compared to wildtype Listeria. The
genetic modification allows the bacteria to invade intestinal epithelial cells and
to cross the murine intestinal barrier with high efficiency. The new bioluminescent
Listeria strain was used to monitor bacterial dissemination in orally infected mice.
For visualization of bioluminescent bacteria during infection we have used the
Xenogen IVIS system, a highly sensitive, low light system optimized for in vivo imaging.
Here, we present first results of different mouse inbred strains (BALB/cByJ,
C57BL/6J, CD1, 129P, C3HeB/FeJ) that behave different in the intensity of infection
and show a time variation regarding bacterial spreading, the crisis of infection
and bacterial clearance. We found CD1 and C3HeB/FeJ mice to be resistant to
orally transmitted listeriosis, they showed reduced bacterial dissemination in the
early phase of infection (during first 72 hrs), and a fast clearance of the pathogen
from day 5 p.i. on. In contrast BALB/cByJ and C57BL/6J mice we found to be more
susceptible after oral infection. This is reflected by slower bacterial clearance and
a reduced survival.
We further give an outlook how we will use this new approach to obtain a detailed
insight in the process of crossing the feto-placental barrier in pregnant mice as
well as the blood-brain barrier after oral infection with our modified Listeria
monocytogenes strain.

AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
Galleria mellonella as model system to study Listeria species-specific
and Listeria monocytogenes serotype-specific pathogenesis
Mraheil, M. A., Krishnendu, M., Hain, T. and Chakraborty, T.
Germany
Essential aspects of the innate immune response to microbial infection are conserved
between insects and mammals. This has generated interest in using insects
as model organisms to study host-microbe interactions. We used the greater wax
moth Galleria mellonella, which can be reared at 37 °C, as a model host for examining
virulence potential of Listeria spp. Here we report that Galleria is an excellent
surrogate model of listerial septic infection, capable of clearly distinguishing
between pathogenic and non-pathogenic Listeria and even between virulent and
attenuated Listeria monocytogenes strains and seotypes. Virulence required listerial
genes hitherto implicated in the mouse infection model, and was linked to
strong antimicrobial activities in both hemolymph and hemocytes of infected larvae.
We conclude that severity of septic infection of L. monocytogenes is primarily
modulated by innate immune responses and suggest the use of Galleria as a relatively
simple, non-mammalian model system that can be used to assess the virulence
of strains of Listeria spp. isolated from a wide variety of settings from both
the clinic and the environment.
Listeria monocytogenes ActA is a key player
in evading autophagic recognition
Pillich, H., Loose, M., Hain, T. and Chakraborty, T.
Germany
Autophagy is a pivotal bulk degradation system that eliminates undesirable molecules,
damaged organelles, and misfolded protein aggregates in response to diverse
stimuli, including infection. Autophagy acts to limit intracellular microbial
growth but intracellular pathogens have evolved strategies to subvert host autophagic
responses for their survival. We found that Listeria monocytogenes ActA,
a surface protein required for actin polymerization and actin-based bacterial
motility, plays a pivotal role in evading autophagy, but in a manner independent of
bacterial motility. We show that L. monocytogenes exploits the biomimetic property
of ActA to camouflage itself with host proteins comprised of Ena/VASP and
the Arp2/3 complex, thereby escaping recognition by autophagy.
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AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
Non-haemolytic and hypovirulent Listeria monocytogenes became
haemolytic and virulent after passage through mice
Secic, I., Lindbäck, T. and Rørvik, L. M.
Norwegian School of Veterinary Science
Two different isolates (from a fish processing plant and a broiler abattoir) showing
typical appearance for L. monocytogenes on OCLA and ALOA, did not express
haemolysis on blood agar with blood from different sources (sheep, horse, bovine,
human) and appeared as L. innocua on RLM. However, biochemical reactions and
16S RNA analysis identified them as L. monocytogenes. High levels (10 9) of each
isolate were injected i.p. into five mice. Three of the mice died after 1-3 days for
both strains. Cultivation from livers and spleens revealed a mixture of haemolytic
and non-haemolytic colonies. Non-haemolytic isolates showed typical appearance
for L. monocytogenes on OCLA and ALOA, but were identified as L. innocua on
RLM. Haemolytic and non-haemolytic isolates from each mouse, as well as the respective
mother strains were identical pulsotypes. In contrast to passage through
mice, fifty passages of the mother strains on RLM did not change the phenotype,
and screening of 10 7 colonies did not reveal any haemolytic subpopulation. HT-
29 cell assay showed that while the non-haemolytic isolates were all hypovirulent,
their haemolytic twins were virulent. Part of the LIPI 1 virulence island of nonhaemolytic
and haemolytic isolates was sequenced. The non-haemolytic isolate
had a seven base insertion in the prfA gene which introduced a stop codon, resulting
in a truncated PrfA protein. The seven base pair insertion was deleted during
infection in mice, resulting in a normal PrfA protein in the haemolytic version.
This study shows that during passage through mice hypovirulent L. monocytogenes
can change to a virulent phenotype. It is not known if this phenomenon may occur
in humans. These strains would not have been recognized as L. monocytogenes unless
OCLA or ALOA had been used as isolation agar.
Mutants of Listeria monocytogenes (Lm) resistant to the polycationic
peptide protamine appear to be attenuated for virulence
Schlech, W.
Dalhousie University, Canada
Previously, we reported the isolation of Lm mutants resistant to protamine, a relatively
inexpensive polycationic peptide derived from salmon and herring milt.
Here, we report the final characterization of two protamine-resistant mutants, emphasizing
their virulence defects, which have led us to believe that protamine-resistant
mutants are attenuated. We also report that p60 mutants, isolated independently
from the protamine-resistance phenotype and previously reported to show
virulence defects, are also resistant to protamine. Protamine-resistant (PtmR) mutants
from the Canadian Maritimes outbreak strain 15U and from the Massachusetts
outbreak strain Scott A, were picked as isolated colonies from TSA plates containing
1 mg/ml of protamine. Electron microscopy and SDS-PAGE were
extensively applied to the initial characterization of the mutants. Swiss-Webster
mice were infected by direct gastric inoculation with different Lm doses and spread
to the spleen and liver monitored at 3 days after inoculation. Survival of Lm in diffusion
chambers surgically implanted in the peritoneal cavity of rats for 3 days, was
also evaluated. By electron microscopy and SDS-PAGE, the PtmR mutants displayed
significant differences in their surface proteins and structure. However,
both showed a reduced ability to infect mice (particularly the liver) and a reduced
survival in the intraperitoneal diffusion chambers. p60 mutants, which are unable
to efficiently infect cells in culture, turned out to also be PtmR. It seems that PtmR
mutants, in spite of their different origin and surface characteristics, are attenuated
for virulence. This possibly represents an example of an inverse fitness-virulence
relationship that could be exploited to select against highly virulent Lm strains in
food products treated with inhibitory concentrations of polycationic peptides.

AREA // B // Listeria monocytogenes as a human and animal pathogen
POSTER PRESENTATIONS // P / 47–81, 182
The role of plasmacytoid dendritic cells in the course of Listeria monocytogenes
infection
Solodova, E., Lienenklaus, S., Jablonska, J. and Weiss, S.
Laboratory of Molecular Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany
Type I interferons (IFNs) play a key role in linking the innate and adaptive arms of
the immune system. There are more that 13 IFN-α and a single IFN-β, all using a
common receptor – IFNAR, which is expressed on wide variety of cell types. Production
of type I IFNs in response to infection with viruses is essential for clearance
of the pathogen from the host. But the impact of these cytokines during bacterial infection
is less defined. Plasmacytoid dendritic cells (pDCs) are known to be major
natural type I IFN producing cells during viral infections, but not much is known
about their impact in the course of bacterial infections. Listeria monocytogenes is
one of the bacteria known to induce type I IFN synthesis. In contrast to viral infections
these cytokines were shown to have detrimental effects for the host during
infection with this bacterium. In our study we used tissue specific conditional reporter
and knock-out mice showing that LysMcre-expressing cells but not pDCs
are responsible for type I IFN production during Listeria monocytogenes infection.
We have also shown that depletion of pDCs revealed a phenotype showing the ability
of pDCs to protect mice infected with Listeria monocytogenes. The aim of my
present work is to investigate this phenomenon und to understand how pDCs are
able to provide protection to mice during Listeria monocytogenes infection.
Oxygen restriction increases the infection potential
of Listeria monocytogenes – a transcriptional analysis
Andersen, J. B., Bergstrøm, A., Knudsen, G., Bak Christensen, B., Ebersbach, T.,
Boye, M. and Rask Licht, T.
Technical University of Denmark, National Food Institute, DTU, Division of Microbiology and Risk
Assessment, Denmark
Listeria monocytogenes has been implicated in several food borne outbreaks as
well as sporadic cases of disease during the last two decades. Increased understanding
of the biology of this organism is important in the prevention of food
borne listeriosis. This is highly relevant for safety assessment of this organism in
food. We have previously shown (Andersen et al., BMC Microbiology; 2007, 7:55)
that the environmental conditions to which L. monocytogenes is exposed prior to
ingestion are decisive for its in vivo infective potential in the gastrointestinal tract
after passage of the gastric barrier. Infection of Caco-2 cells revealed that Listeria
cultivated under oxygen-restricted conditions were approximately 100 fold more
invasive than similar cultures grown without oxygen restriction. This means that
not only the number of Listeria present in a given food item, but that also the physiological
condition of these bacteria is important for food safety. The in vitro and in
vivo data suggest that an oxygen-restricted L. monocytogenes cell represents a significantly
higher risk than a cell grown without oxygen restriction. In order to
identify transcriptional differences contributing to different invasiveness, microarray
gene chip technology was applied to cDNA created from RNA isolated
from oxygen restricted and non-restricted cultures. The analysis confirmed several
relevant genes to be differentially transcribed in the two environmental conditions
e.g. genes related to virulence potential of L. monocytogenes.
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AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Semi-automated repetitive sequenced-based PCR compared to pulsed
field gel electrophoresis for Listeria monocytogenes sub-typing
Roussel, S., Félix, B., Vignaud, M.-L., Tam Dao, T., Marault, M. and Brisabois, A.
AFSSA, France
Listeriosis is a severe infection which mainly impacts pregnant women, neonates
and immuno-compromised adults. The commercially available, semi-automated
rep-PCR assay system, DiversiLab, has been successfully used for subtyping several
species of bacteria. We compared here the DiversiLab System with macrorestriction
analysis by pulsed field gel electrophoresis (PFGE) which is currently the gold
standard for molecular subtyping of Listeria monocytogenes. We used a panel of
116 human and food L. monocytogenes strains for the comparative evaluation.
Among these strains, there were 4 pairs of duplicates; 13 strains were epidemiologically
related and the remaining food isolates were epidemiologically unrelated.
The strains of different serotypes revealed distinct ApaI-PFGE types, Rep-PCR
types (RT) and AscI- PFGE types except for one RT and one AscI- PFGE type. The
four doublets displayed a unique RT, AscI and ApaI PFGE pattern showing the
good reproducibility of the three methods. The epidemiologically related strains
were clustered in the same RT and PFGE types. The Simpson’s index of diversity
was 0.954; 0.988; 0.994; 0.998 for DiversiLab, AscI-PFGE, ApaI-PFGE and
AscI/ApaI-PFGE respectively. Thus, PFGE was more discriminating than DiversiLab.
However, for 1/2a serotype strains, six AscI-PFGE and three ApaI-PFGE
types were divided into different RT. DiversiLab allowed a good discrimination
between serotype 1/2a strains. This investigation also demonstrated the ability of
the DiversiLab to differentiate serotypes 4b and 1/2b strains. DiversiLab is less
labour-intensive than PFGE and provides results in less than 24 hours compared
with 30 hours to 3 days for PFGE from the time a pure culture of the bacteria is obtained.
Based on these results, DiversiLab may be useful for tracking the source of
contamination in food processing facilities and their environments.
Listeriosis: a frequent cause of fatal encephalitis in France
with high case fatality
Mailles, A. 1, Vaillant, V., Lecuit, M. 2 and Stahl, J.-P. 3
1. Institut de Veille Sanitaire, France
2. Institut Pasteur, France
3. University Hospital of Grenoble, France
In 2007, we carried out a national prospective study in 106 hospital units to assess
the aetiology, clinical patterns and outcome of infectious encephalitis in
France. Among 253 case-patients, encephalitis due to L. monocytogenes was identified
in 13 people. A case of encephalitis was a patient aged at least 28 days, hospitalised
in France with an acute onset of illness and at least one abnormality of
the CSF, and fever and decreased consciousness or seizures or altered mental status
or focal neurological signs. All case-patients had a complete biological investigation
to assess the etiologic diagnosis. Clinical data were collected using standardised
questionnaires. Listeriosis case-patients were compared to other
case-patients enrolled in the study. Thirteen case-patients were identified with
encephalitis due to L. monocytogenes among 253 case-patients (5 %) and 131 casepatients
with an etiological diagnosis (10 %). Listeriosis was the fourth most frequent
cause of encephalitis identified after HSV (n=55), VZV (n=20) and tuberculosis
(n = 20). Listeria was isolated in CSF from XX patients, from blood only from
1 case. XX cases had a positive PCR on CSF. The last patient was diagnosed on a
combination of clinical and epidemiological criteria. Eleven listeriosis case-patients
were confirmed cases, 1 was a probable case and 1 was a possible case. Their
mean age was 71 years and 9 were men. Listeriosis case-patients were more likely
to have a current treated cancer (23 % vs 5 %, p=0.005) and to present with cranial
nerves impairments (38 % vs 17 %, p=0.02). Six of 13 (46 %) listeriosis casepatients
had a fatal outcome vs 20/216 (8.5 %) of other patients (p=0.007). These
results showed that L. monocytogenes is a frequent cause of encephalitis in France
and confirmed its severity. Listeriosis should be considered early in the course of
encephalitis, as specific and efficient treatment can be proposed.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Listeria monocytogenes: identification and subtyping
Favretti, M.
Istituto Zooprofilattico Sperimentale delle Venezie, Italy
Listeria monocytogenes is a common environmental organism and an important
food-borne pathogen too. Pregnant women, neonates, elderly and immunocompromised
patients are at greatest risk of acquiring listeriosis. Since the recognition
of Listeria monocytogenes as a food-borne pathogen there have been rapid advances
in the development of suitable methods for isolation and identification.
Although all Listeria monocytogenes strains are considered pathogen, only some
serotypes are predominant and frequently involved in outbreaks. So it is very important
to obtain viable isolates for typing, elucidating outbreaks and tracing the
source of infection. The differentiation of Listeria monocytogenes isolates with
phenotypic or molecular subtyping methods has provided useful information related
to the phylogeny, epidemiology and ecology of the organism and also has permitted
the correlation of isolates from food and from human case in order to underline
any relationship between them. Although there are several techniques for
identification and typing available at the moment, it is not possible to identify the
“gold standard” method yet. Therefore, using in parallel different methods ensures
a correct and complete identification of strains. We present an overview of
the techniques used to isolate, to identify and to subtype Listeria monocytogenes;
the various subtyping systems provide different degrees of discrimination among
isolates. Subtyping methods for Listeria monocytogenes include phenotypic (e.g.
serotyping and phage-typing) and different DNA–based subtyping methods (e.g.
multilocus enzyme electrophoresis, ribotyping, pulsed-field gel electrophoresis
(PFGE), polymerase chain reaction (PCR)).
Virulotyping of Listeria monocytogenes by high resolution melt analysis
Amar, C. F. 1, Tamburro, M. 2, Dear, P. 3 and Grant, K. 1
1. Health Protection Agency, Centre for Infections, UK
2. University of Molise, Campobasso, Italy
3. Laboratory of Molecular Biology, Cambridge, UK
L. monocytogenes causes a severe foodborne infection in vulnerable people. Isolates
are known to vary in their ability to cause infection but current typing methods
do not provide information on strain pathogenicity. Six key L. monocytogenes
virulence genes, clustered together on pathogenicity island, LIPI-1 (9Kb), are
known to be essential for intracellular survival, whilst two other virulence genes,
internalin A and B, are responsible for host cell invasion. Many food isolates produce
truncated internalin proteins due to point mutations and thus have reduced
pathogenicity. Detection of these mutations and those in other virulence genes
are likely to provide valuable information on strain pathogenicity. High Resolution
Melt (HRM) analysis is a powerful technique capable of identifying sequence variations
in PCR amplicons by accurately determining their melting temperature
(Tm). This technique was used to generate virulence fingerprints, using 81 markers
spanning LIP1 and Internalin A and B genes. Strains of L. monocytogenes from
human cases, food and the environment were compared with results generated
using L. monocytogenes EGDe. Considerable variations in Tm were found across
markers for LIP1 and internalin A and B genes which were confirmed by sequencing.
Eight markers had Tms specific to genetic lineage I and 11 to genetic lineage II.
Fifteen markers had conserved sequences across both lineages with those spanning
the hlyA gene being the most conserved. In the housekeeping gene, prs,
marker variations were only detected in serotype 4b strains. Tm variations were
detected in markers for actA, intA and intB genes in which point mutations are
associated with the expression of truncated proteins and a concomitant reduction
in virulence. HRM is an accurate and rapid technique for detecting sequence diversity
within bacterial genes and not only presents a novel method for strain characterisation
but also offers a unique potential to inform on strain pathogenicity.
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Risk factors for nonperinatal listeriosis mortality
in Los Angeles County, California, 1992–2004
Guevara, R. E.*, Mascola, L. and Sorvillo, F.
County of Los Angeles Department of Public Health, USA
Listeriosis is a relatively rare foodborne disease but has significant public health
implications as it has a case-fatality rate of 20 % and it causes 28 % of all foodborne
disease related deaths. Yet, data on the risk factors for listeriosis mortality
have been limited. A 13-year retrospective cohort study was performed to describe
nonperinatal listeriosis mortality in Los Angeles County, California, during
1992–2004. A nonperinatal listeriosis case was defined as a non-pregnant person
>42 days old with a positive culture of Listeria monocytogenes and residence in
Los Angeles County. Causal modeling based on prior knowledge and bivariable
analyses was performed to determine a comprehensive unconditional multivariable
logistic regression model with 29 main effect variables. With 281 nonperinatal
listeriosis cases, multivariable analysis found non-hematological malignancy
(odds ratio: 5.92, 95 % confidence interval: 1.85-18.9), alcoholism (4.63, 1.36-15.8),
age ≥ 70 years (3.44, 1.50-7.87), steroid medication (3.34, 1.38-8.08), and kidney
disease (2.94, 1.18-7.31) to be statistically significant risk factors for mortality.
Other listeriosis mortality risk factors with adjusted odds ratios >1.5 included
blood transfusion, asthma, black race-ethnicity, Asian race-ethnicity, antibiotics,
hypertension, chemotherapy, and Hispanic race-ethnicity. Cases admitted with
sepsis only had the highest mortality (23.7 %), while cases with meningitis only
had the lowest mortality (3.13%). The findings of this study should highlight the
importance of certain underlying risk factors and their association with nonperinatal
listeriosis-related deaths. (This study was published in Clinical Infectious
Diseases 2009; 48:1507-15 ©2009 by the Infectious Diseases Society of America.
http://www.journals.uchicago.edu/loi/cid).
* Participation Supported by IUFoST.
Molecular typing of Listeria monocytogenes isolated from ovine sausage
Nives, M. R. 1, Mele, P. 1, Parisi, A. 2, Latorre, L. 2, Virgilio, S. 1 and Tola, S. 1
1. Istituto Zooprofilattico Sperimentale of Sardinia, Italy
2. Istituto Zooprofilattico Sperimentale of Apulia and Basilicata Italy
Sardinia, an island located in the middle of Mediterranean sea, has about four million
of milking sarda sheep. In recent years in addition to milk and cheese production
other products from sheep meat have been commercialized (ham and
sausage). In this survey, between 2007 – 2008, five sampling were performed inside
a factory located in the northern side of Sardinia. We monitored the different
stages of sausage production for detecting the presence of Listeria monocytogenes
in food matrices, the equipment and the environment. Isolation and identification
of L. monocytogenes were carried out according to ISO 11290-1/2:1996/98
amendment 1-2004. The isolates were characterized using serotyping and three
different molecular methods: PFGE, MLST, VNTR according to previous published
protocols. Moreover five virulence genes (actA, hlyA, plcB, prfA, iap) were detected
by PCR. L. monocytogenes was isolated in all the phases of the sausage production
(raw minced meat, meat mixture after salt and spice addition, sausage after desiccation
time, sausage during different seasoning periods) and from the floor drain.
On the whole 21 out of 126 samples resulted contaminated. The L. monocytogenes
count resulted in all the samples higher than 100 UFC/g (Regulation EC n°
2073/05). All isolates resulted PCR-positive for the investigated virulence genes.
PFGE, MLST and VNTR identified two different molecular profiles. Five isolates,
marked as type A and belonging to the serotype 1/2a, were isolated just from one
batch samples whereas the type B (n=16), belonging to the serotype 1/2b, was isolated
from three different food batches and from the floor drain. Our results
demonstrate an high occurrence of L. monocytogenes in the studied facility. So the
sheep sausage production must be object of a careful and constant vigilance activity
and the isolates should be characterized using molecular methods for timely
monitoring the contaminations and to play out the due prevention actions.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
A novel phage-PCR assay for the rapid detection of viable Listeria monocytogenes
in food within 24 h
Elemam, M. M.
School of Biosciences, University of Nottingham, UK
Listeria monocytogenes is a pathogen of public health significance causing occasional
outbreaks and sporadic cases of foodborne illness. This microorganism is of
particular concern in the food industry due to its widespread distribution in nature,
its ability to survive in a wide range of environmental conditions, and its ability
to grow at refrigeration temperatures. The standard microbiological method
for L. monocytogenes detection in foods (ISO 11290-1/ A1 - 2004 and NHS-2009) is
costly and time consuming; require at least 6 days obtaining a results. The development
of a rapid and reliable test capable of detection very low numbers of the organism
in ready to eat products is of significant importance to the food industry.
The Phage Amplification assay has been developed as a rapid method for the detection
of L. monocytogenes. We have been using the broad host range phage A511
to develop such a method for detection of Listeria monocytogenes in both cheese
and stomached food samples. Successful development of the assay requires a virucide
to achieve differential inactivation of the phage without affecting the viability
of the target cell to be detected. Several different chemicals were evaluated as potential
virucides, but tea extracts were found to be the most effective virucidal
agent. The efficacy of the new assay was tested using cheese samples and was to
shown to be able to detect low numbers of cells, in only 24 h in compared to the 6
days to conventional culture methods. Because of the broad host range of the
phage the identity of the cell detected must be confirmed using PCR amplification
of signature sequences from the phage plaque and several different PCR
strategies have been evaluated. The combined phage-PCR method would allow
rapid screening for food products prior to release from the factory.
Perinatal listeriosis in Los Angeles County, California, 1992 – 2004
Guevara, R. E.* and Mascola, L.
County of Los Angeles Department of Public Health, USA
In Los Angeles County (LAC), California, physicians and laboratories are required
to report all listeriosis to the LAC Department of Public Health. From this passive
surveillance, a population-based epidemiologic study was performed to further describe
perinatal listeriosis. Perinatal listeriosis was defined when Listeria monocytogenes
was identified from a normally sterile site between a mother-fetus or
mother-infant (infant age < 43 days old) pair. During 1992 – 2004, of 413 listeriosis
cases reported, 122 (30 %) were perinatal listeriosis cases. Perinatal listeriosis had
a higher average annual incidence rate (5.9 cases/100,000 live births/year) and
mortality (32.2 %) than nonperinatal listeriosis (2.5 cases/million people/year, and
18.6 %, respectively). Disease onset during pregnancy defined intrapartum cases
(n=93, 76 %), during 0-6 days after birth defined early onset cases (n=21, 17 %), and
during 7-42 days after birth (n=8, 7 %) defined late onset cases. For intrapartum,
early-onset, and late-onset cases, fetal/infant mortality was 36.6 % (n=34), 14.3%
(n=3), and 0%, respectively. No maternal deaths occurred. Median gestational age at
admission of intrapartum cases was 28 weeks (range 10-40 weeks) with maternal
symptoms commonly including fever (84 %), abdominal pain (35 %), chills (29 %),
back pain (24 %), and headache (20 %). Intrapartum listeriosis cases had maternal
infections of both sepsis and meningitis (n=2, 2.2 %) and sepsis only (n=72, 77.4 %),
but mothers with symptoms in 19 (20.4 %) intrapartum cases were culture-negative
or not cultured. Among intrapartum cases with hospital admission for listeriosis
before 38 weeks gestation, 33 (35 %) had fetuses continue into pregnancy for
more than 7 days (6 fetal demises/stillbirths with median of 13.5 days after onset, 4
sick newborns with median 30.5 days after onset, 17 healthy newborns with median
25 days after onset, and 6 unknown birth outcomes). Although annual incidence
of perinatal listeriosis has been decreasing, opportunities such as culturing
symptomatic mothers for L. monocytogenes exist to further prevent perinatal listeriosis
morbidity and mortality.
* Participation Supported by IUFoST.
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AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Antimicrobial resistance of Listeria monocytogenes human strains
isolated since 1926 in France
Morvan, C., Moubareck, A., Leclercq, M., Herve-Bazin, S., Bremont, M., Lecuit, P.
and Le Monnier Courvalin, A.
Institut Pasteur, France
Amoxicillin plus gentamicin is the treatment of choice for listeriosis, a foodborne
infection caused by Listeria monocytogenes (Lm). Lm is considered universally
susceptible to antibioticsactive against gram-positive bacteria, but resistant strains
have been reported. Lm is considered universally susceptible to antibiotics active
against Gram-positive bacteria except 3 rd generation cephalosporins, but resistant
strains have been reported. We have studied retrospectively the antimicrobial resistance
of Lm human strains isolated between 1926, the year of its discovery, and
2007. 4 816 human Lm strains were picked randomly in the strain collection of the
WHO collaborating for Listeria. Their susceptibility to 23 clinically relevant antibiotics
was determined by disc diffusion. MICs of fluoroquinolones and penicillins
were determined by E-test. All resistant strains were typed by PFGE and
those displaying a unique profile were screened for the presence of antibiotic resistance
genes by PCR. The mechanisms leading to detected resistance was studied
for each resistant strain. Sixty-one human strains (1.27 %) were resistant to one
or more antibiotics. Resistance to the tetracyclines (n = 34) and fluoroquinolones
(n = 20) was more common and emerged since the late 80s and the late 90s, respectively.
Resistance to fluoroquinolones was attributed to active efflux. Resistance
to the tetracyclines was encoded by tet(M), presumably carried by a conjugative
transposon in the 14 strains which also harboured the int gene. We also
detected the first human isolate with high-level resistance to trimethroprim, due
to the dfrD gene. Although no penicillin-resistant strain was identified, MICs have
significantly increased since 1926 with values up to 2 µg/mL. In conclusion, L.
monocytogenes has not acquired significant clinically-relevant antibiotic resistance.
Importantly, no resistance to amoxicillin and gentamicin, the recommended
antibiotic regimen for listeriosis, was found. Yet, significant increase of penicillin
MICs and the description of a strain resistant to trimethoprim reinforce the need
for surveillance and are in favour of systematic antibiotic susceptibility testing including
penicillin MICs determination.
Today and tomorrow: The molecular epidemiology of listeriosis in the UK
Grant, K., Amar, C., Matos, J., Mook, P., Little, C. and Gillespie, I.
Health Protection Agency, UK
Listeriosis is a rare but severe foodborne infection caused by Listeria monocytogenes:
in the UK it is the leading cause of death due to a foodborne pathogen. Since
2000 there has been an increase in the number of cases associated with patients
aged > 60 years presenting with bacteraemic infection in the UK and in other European
countries and, yet, the reason for this increase remains to be established. In
the UK, the number of pregnancy related cases of listeriosis has remained relatively
stable over the past few years, although, more recently there has been a noticeable
increase in the proportion of cases describing themselves as of ethnic origin.
Particularly concerning is that since the beginning of 2009, there has been an
increase in the total number of pregnancy related cases. Because the incubation
period for listeriosis is long and those at risk of infection are often ill with serious
underlying conditions the investigation of individual cases and outbreaks is often
difficult with an increased emphasis on microbiological evidence. Approximately
170 human and 900 food Listeria monocytogenes isolates from across England and
Wales are received annually by the Laboratory of Gastrointestinal Pathogens. All
isolates are identified and characterised using molecular methods including PCR–
based serotyping and amplified fragment length polymorphism. This data is used
in conjunction with standardised epidemiological data to confirm and link sporadic
cases; to identify and investigate clusters and outbreaks; to provide information
on routine food testing as well as for national surveillance purposes such as
identifying risk exposures for human infection. This paper will demonstrate, with
recent examples from the UK, the contribution of molecular typing to epidemiological
investigations and will outline a new direction for typing Listeria monocytogenes
which will not only be discriminatory but provide additional information
on the relatedness of strains and their pathogenic potential.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Use of Fluorescent Amplified Fragment Length Polymorphism
for improved typing of Listeria monocytogenes
Matos, J., Amar, C., Desai, M., Cross, L. and Grant, K.
Health Protection Agency, UK
Listeria monocytogenes is a ubiquitous, psychrotrophic, Gram positive foodborne
pathogen which has the ability to persist in food processing environments and
grow in a range of ready-to-eat foods. It causes the disease listeriosis which mainly
affects those with a weakened immune system including pregnant women,
neonates, the elderly and immunocompromised individuals. Although a rare disease
it has a high mortality rate (20 – 30 %) and is currently responsible for more
deaths than any other foodborne pathogen in the UK. Typing of L. monocytogenes
strains is critical to investigating both sporadic cases and outbreaks enabling the
identification of the food vehicle and tracing the origin of contamination. The success
of any typing method depends on its discriminatory ability, reproducibility,
cost of the procedure and speed to getting results. At present Pulse field Gel Electrophoresis
is generally accepted as the gold standard typing method for epidemiological
investigations involving L. monocytogenes. However, it is not without its
drawbacks including being lengthy and labour intensive to perform, making it unsuitable
for real time analysis. PFGE also suffers from a lack of interlaboratory
reproducibility and the analysis of profiles is open to subjective interpretation.
This study investigated the use of Fluorescent Amplified Fragment Length Polymorphism
(FluAFLP) for typing 842 isolates of L. monocytogenes from clinical
cases, foods and food processing environments using two restriction enzymes
(HindIII and HhaI). All isolates were previously subtyped by multiplex PCR-based
serotyping and a 133 subset had also been previously typed by PFGE using AscI.
fAFLP was found to be highly discriminatory (D.I. = 0.987), rapid to perform, reproducible
and readily amenable to automation. Cluster analysis of isolates
showed that fAFLP efficiently separated lineage I isolates from those of lineage
II and that there was a high level of interlineage discrimination.
Towards an application for field samples of a multipathogen platform
for molecular detection of raw milk pathogens
Omiccioli, E1, Amagliani, G. 2, Brandi, G. 2, Tonucci, F. 3, Foglini, M. 3 and Magnani, M. 2
1. Diatheva s.r.l., Italy
2. Department of Biomolecular Science, University of Urbino, Italy
3. Istituto Zooprofilattico Sperimentale Umbria e Marche, Italy
Listeria monocytogenes, together with Salmonella spp. and Escherichia coli O157,
are pathogens associated with various food categories, included raw milk, which
commercialisation for human direct use has been encompassed by the
853/2004/EC. Since this product can constitute a risk for human health, the absence
of such bacteria should be assessed by sensitive and specific diagnosis. Molecular
diagnostic tests in multiplex format proved to be useful for the rapid identification
of food contaminations caused by more than one microbial species. As
part of a European Research project (DIAGNOSIS), we developed an enrichment
multiplatform Real-Time PCR, including an internal amplification control ensuring
the reliability of results, for the detection of all three pathogens. The assay
combines an enrichment step in a medium properly formulated for the simultaneous
growth of target pathogens (Multipathogen enrichment medium), a DNA
isolation method, and a multiplex Real-Time PCR detection system based either on
dual-labelled probes (MultipathogenFLUO), or on melting curve analysis (MultipathogenHRM).
The entire system enables the detection of as few as 1 CFU of each
pathogen in 125 ml milk in only two working days and it is now available from
Diatheva srl, Fano, Italy. An application study for field samples is currently in
progress. A local dairy farm, licensed to the direct selling of raw milk through automatic
distributors, was selected for the collection of raw milk samples and swabs
from filters located between milking rooms and storage tanks. Samples are
analysed by the official reference ISO methods and also culture-enriched in the
Multipathogen enrichment medium. Aliquots from all enriched cultures are subjected
to DNA column-based extraction and PCR amplified by both the MultipathogenHRM
and MultipathogenFLUO kits. The comparison of results will allow
the estimation of method equivalence and the compliance of the molecular kits
with law provisions, contributing to promote the adoption of PCR-based methods
alongside the traditional diagnostic procedures.
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AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Detection and recovery of Listeria species from stainless steel
Boone, R., Iugovaz, I, Trottier, Y.-L. and Pagotto, F.
Health Canada
The 2008 Canadian listeriosis outbreak in ready to eat meat raised many issues
over the safety and reliability of the national food supply, including the reliability
of detection methodologies aimed at food-contact surfaces made of stainless steel.
Five methods pertaining to the recovery of Listeria species including L. monocytogenes
from stainless steel were assessed. A cocktail of L. monocytogenes (serovars
1/2a, 1/2b, 4b), L. innocua, plus a Gram-positive organism (Staphylococcus aureus)
was inoculated at different levels onto stainless steel. Inocula were recovered using
MFLP-41 and assessed using different detection methods, including Petrifilm
(3M), BAX Genus Listeria (Qualicon), BAX Listeria monocytogenes, and four
VIDAS kits (BioMérieux) (LDUO, LIS, LMO2 and LSX). Direct plating onto selective
and chromogenic agars were done simultaneously from each broth and each
kit was compared to the “gold standard”, MFHPB-30. Experiments using skim
milk powder assessing cell viability revealed a consistent 1-log die-off after 24 h.
The VIDAS strips DLIS, LIS and LSX had 74 positive samples out of 84 tested following
52 hours of incubation in LX Broth while the BAX Genus Listeria had 72
positive samples out of the 82 tested. The VIDAS strips DLMO and LMO2 yielded
52 and 51 positive samples out of 79 tested, respectively; whereas the BAX L. monocytogenes
detected 50 of 81 samples tested. Petrifilm modifications to increase incubation
time from 30 h to 48 h are being suggested due to 27 out of 84 samples
were negative at 30 h but positive at 48. Differences amongst methods were noted
at the single cell inoculum level. None of the methods had false positive results.
Through the study of different rapid detection methods, options can be brought
forward to improve and ensure optimal conditions for Listeria detection (and recovery)
from stainless steel environments.
Natural carriage of Listeria in fresh-water fish in Russia
Egorova, I. 1, Voronin, M. 2, Selyaninov, Y. 1 and Kolbasov, D. 1
1. National Institute of Veterinary Virology and Microbiology Pokrov Russia
2. National Park “Zavidovo”, Russia
To study the extent of Listeria natural carrier stage in fresh-water fish, the
Ivankovo water storage reservoir was observed. In the period of 2006 to 2008, 642
fish specimens belonging to 18 fresh-water fish species were examined. In total, 34
listerial cultures of monocytogenes and innocua species were isolated in fish, 35,3 %
of them belong to L. monocytogenes. Listeria isolation rate was 5,38 %. L. monocytogens
were isolated predominantly from plant-feeder fish like bream, crucian
carp and silver bream. L. innocua - in bream, pike, crucian carp, redeye and perch
of all age groups. Listeria carriers were found most often in areas where animal
farm buildings and treatment facilities were located. The primary Listeria locations
in fish were muscle tissues, gill rakers and parenchymatous organs. In two
cases L. innocua were isolated from fresh-water fish intestines. The peak of Listeria
carrying in fish falls on summer periods (40 %). In autumn and winter periods
the detection rates were 28 %. In spring the index dropped to 4 %. All the cultures
are quite typical specimens of their species. L. monocytogenes cultures produced
hemolysins and phospholipases, were agglutinated with specific sera and lysed
with phage L2A, and induced purulent conjunctivitis in guinea pigs. L. innocua
cultures were hemo- and phospholipase-negative and were agglutinated with a
serum of serogroup II, were lysed with phages L4A and did not induce mice death
due to intraperitoneal inoculation at a dose of 10 9 m.b. The experimentally determined
diversity of the of Sma I pulse electrotypes suggests there are both associated
and independent Listeria reservoirs and there is a circulation of several clone
variants of the pathogen in the fresh-water fish population.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Prevalence of Listeria in wild fauna in Russia
Egorova, I. 1, Fertikov, V. 2 and Kolbasov, D. 1
1. National Institute of Veterinary Virology and Microbiology Pokrov Russia
2. National Park “Zavidovo”, Russia
Monitoring of Listeria in environmental objects and also among ungulate wildlife
inhabiting forest hunting ranges of the Central region of Russia has been carried
out. 662 samples of soil from feeding grounds, decaying plant residues, forages
and wildlife faeces, and also brain, muscle tissue and/or parenchymal organs of
the axis deer, the maral, the elk and the wild boar, were tested and 68 listerial cultures
were found. The organism carriage indices for the axis deer, the maral and
the wild boar in the areas of Tver, Moscow and Kaluga regions were at a similar
level making up 2.0 to 2.7 %. In Vladimir region the indices turned out to be a little
higher gaining 12 % for the wild boar and 5.45 % for the axis deer, which is most
likely due to some differences in the forms of economic activities and inadequate
observance of sanitary-and-hygienic requirements. Examinations of elk faeces revealed
Listeria in none of the cases which is probably explained by specificity of
the elk nutrition. Examinations of environmental objects showed that all of them
were to a variable extent contaminated by Listeria of both pathogenic and nonpathogenic
species. Listeria were mainly isolated in soil samples taken from feeding
grounds used for seasonal feeding of the wildlife. In samples from wild animals
Listeria were found in two cases in axis deer brain and lymph node, and also
in boar liver and lung. The isolate taken from the axis deer is of a certain research
interest, as according to all the phenotypic indications it was classified to L. innocua,
although investigation of its genetic structure showed presence of pathogenicity
genes typical for pathogenic Listeria species in its genome. The remaining
isolates of Listeria found in the course of the monitoring investigations belonged
basically to two species, namely L. monocytogenes and L. innocua.
Quantitative assessment of the exposure to Listeria monocytogenes
from soft-ripened cheese consumption in North America: a joint
FDA/Health Canada project
Gendel, S. 1, Pouillot, R.1, Murray, C. 1, Farber, J. 2, Ross, W. 2, Couture, H. 2 and Jean, A. 2
1. Food and Drug Administration, USA
2. Health Canada
The United States and Canada continue to experience sporadic illnesses and outbreaks
of listeriosis associated with the consumption of cheese, particularly soft
and soft-ripened cheese. Therefore, the US Food and Drug Administration and
Health Canada jointly conducted a risk assessment to assess the public health impact
of Listeria monocytogenes in soft-ripened cheese in the US and Canada, and to
evaluate the impact of current and potential mitigation and control strategies. To
support this risk assessment, a probabilistic model was developed to assess
changes in L. monocytogenes prevalence and levels from “farm to fork” in multiple
scenarios. This model evaluated the impact of contamination of the milk used for
cheese making, in-plant environmental contamination, partitioning, mixing, bacterial
growth and bacterial inactivation though the complete product pathway.
Model calculations and parameters for a base model were derived from data in the
published literature and from specific databases from both countries. The general
framework of the model was a second-order simulation that allowed separate evaluation
of variability and data uncertainty. An original back-calculation was used to
evaluate the frequency and level of environmental in-plant contamination based
on published data on L. monocytogenes prevalence and levels in soft-ripened
cheeses at retail in the US. This back-calculation model was integrated using the
Monte-Carlo method. The results of the back calculation suggest that in-plant
contamination is infrequent and generally leads to a low levels of L monocytogenes
per cheese (< 25 cfu in each whole 250 g cheese). Because data on production levels
and practices for the cheese making industry are limited, the model was used to
compare exposure estimates obtained using the base model to estimates obtained
using alternate scenarios. For example, scenario analysis suggested that, for contaminated
cheese, the level of exposure is highly sensitive to storage conditions.
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AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Human listeriosis in England, 2001-2007: association with neighbourhood
deprivation
Gillespie, I. A., Mook, P., Little, C. L., Grant, K. and McLauchlin, J.
Health Protection Agency, UK
Listeriosis is a rare but severe disease affecting mostly the elderly and the immunocompromised,
and, to a lesser extent, pregnant women, unborn or newborn
infants. Despite a high mortality rate, the socioeconomics of listeriosis has not
been studied in detail, meaning that health inequalities which might exist in relation
to this disease are not apparent. Laboratory surveillance data on cases of Listeria
monocytogenes infection reported in England between 2001 and 2007 were
linked to indices of deprivation and denominator data using patients’ postcode.
Incidence relative to increasing quintiles of deprivation was calculated by fitting
generalized linear models whilst controlling for population. Patient exposure data
were scrutinised and compared with commercial food purchasing denominator
data to further quantify the risk. For all patient groups, listeriosis incidence was
highest in the most deprived areas of England when compared to the most affluent,
and cases were more likely to purchase foods from convenience stores or from
local services (bakers, butchers, fishmongers and greengrocers) than the general
population. Patients’ risk profile also changed with increasing neighbourhood deprivation.
With increased life expectancy and rising food prices, food poverty could
become an increasingly important driver for foodborne disease in the future.
Whilst Government policy should continue to focus on small food businesses to
ensure sufficient levels of food hygiene expertise, tailored and targeted food safety
advice on the avoidance of listeriosis is required for all vulnerable groups. Failure
to do so may enhance health inequality across socioeconomic groups.
Characterisation of antibodies for use in an antibody-based sensor for
on-site detection of L. monocytogenes
Gilmartin, N., Hearty, S. and O’ Kennedy, R.
School of Biotechnology and National Centre for Sensor Research, Dublin, Ireland
L. monocytogenes is a facultative anaerobic, non-sporing, Gram-positive, motile,
rod-shaped bacterium and is an important food-borne pathogen. It is a major
causative agent of listeriosis, an infection which can kill vulnerable people such
as the elderly, newborns, pregnant women and people suffering from immunocompromising
diseases. The current methods for sampling and detection of L.
monocytogenes can result in extensive treatment times, limited sensitivity and
very low recovery rates of the microorganism (due to the formation of biofilms).
Antibody-based sensors permit the rapid and sensitive analysis of a range of
pathogens and associated toxins. They offer many advantages such as improved
sensitivity, real-time analysis and quantification. An anti-L. monocytogenes monoclonal
antibody (2B3) was isolated from a panel of hybridomas produced using L.
monocytogenes serotype 1/2a as the immunogen. This antibody reacted with L.
monocytogenes serotypes 1a, 1/2a, 1/2b, 1/2c, 3a, 4a and 4b but did not react with related
Listeria spp. and non-Listeria spp. The mAb was further evaluated in both
enzyme-linked-immunosorbent assay (ELISA) and fluorescence-linkedimmunosorbent
assay (FLISA)-based formats and in a surface plasmon resonance
based biosensor platform. This antibody will be exploited for use in an antibodybased
sensor as part of the BIOLISME project which aims to develop a system to
monitor the contamination levels of L. monocytogenes in industrial food-producing
plants. Preliminary work on the use of this antibody in the detection of L. monocytogenes
in biofilms will also be presented.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Food Investigation of sporadic cases of neuroinvasive listeriosis
Goulet, V. 1, Leclercq, A. 2, Laurent, E., King, L., Dusch, V., Salem, S., Vaillant, V. 1,
Chenal-Francisque, V. 2, de Valk, H. 1 and Pihier, N. 3
1. Institut de Veille Sanitaire, France
2. National Reference Centre and WHO collaborating centre for Listeria, France
3. Direction générale de l’Alimentation, France
Listeriosis in France is mandatory notified. For each case, a detailed food history is
collected. For cases with central nervous system involvement who consent, samples
are taken from foods and the environment in the kitchen of their residence or
of hospital for patients hospitalized before disease onset. We present results of
investigations 2003-2008. Samples are taken by officers of inspection services of
the ministry of agriculture, within 14 days after diagnosis of the case, and sent to
their authorised laboratories. Strains of Listeria monocytognes (Lm) isolated from
these samples are sent to the National Reference Centre who performs PCR
serotype, and PFGE (Apa1 and Sma1) typing of the food and patient isolates. Lm
contaminated food is traced back with further investigations at the production
sites. 140 cases have been investigated. Lm was isolated in 63 (45 %) investigations,
from cheeses, meat products, and the environment. For 23 investigations,
the food or environmental strains had the undistinguishable PFGE profiles of the
patient strain. At 2 occasions, the patient strains were isolated from the kitchen
environment in their hospital. In 3 other investigations, the patient strain was also
isolated at the production site of the contaminated food. At one occasion, the early
investigation of a case later shown to be part of a cluster, allowed the prompt investigation
in a cheese producing factory and the confirmation of the source of
the outbreak. Food investigation of listeriosis cases are of interest because they
can lead to control measures in contaminated production sites and limit the size of
outbreaks of listeriosis by rapidly identifying their sources. However the yield of
these investigations is limited because numerous unpacked food products in the
patient’s refrigerators can not be easily identified. An important finding is that
Lm strains can colonise the environment of hospital kitchens.
Evaluation of the antimicrobial activity of Vaccinium myrtillus, Prunus
domestica and Myrtus communis against Listeria monocytogenes
Serio, A., Di Pasquale, F. and Paparella, A.
Department of Food Science, University of Teramo, Mosciano Stazione (TE), Italy
Essential oils and plant extracts have been increasingly used as natural food
preservatives. They are considered consumer-friendly and can be used without
any specific authorization or labelling change. In this study, three aqueous plant
extracts were evaluated for their antimicrobial activity against 45 Listeria monocytogenes
strains. Among blueberry, plum and myrtle extracts, the latter was the
most effective against the strains isolated from smoked salmon and meat products,
with MIC (Minimal Inhibitory Concentration) values generally ranging from
0.5 to 2.0 %. Interesting results were also obtained with the plum extract, although
with MIC values from 1 to 4 %. Blueberry was effective only at high concentration
levels, even above 10 %. Isolates from the smoked salmon industry were generally
less sensitive, suggesting that resistance to plant extracts may somehow be related
to strain origin. A 3-factor-5-level Central Composite Design was used to evaluate
the extracts activity in combination with compounds commonly present in some
food products, where they can constitute a hurdle for bacterial growth. Myrtle extract
activity was boosted by lactic acid and salt, while ATCC7644 strain, used as
control, was particularly susceptible to all combinations. These results are in line
with our previous research on volatile compounds, where L. monocytogenes was
inhibited at low extract concentrations, without significantly affecting food flavor.
Further studies will be carried out to evaluate the potential of myrtle extract in
food biopreservation.
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AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
High throughput quantitative detection of Listeria monocytogenes
in food by RealTime PCR
Cammà, C., Ancora, M., Rizzi, V., Sperandii, A., Prencipe, V. and Migliorati, G.
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy
Listeria monocytogenes is one of the most important food-borne pathogens and
can cause a severe disease in human. The Commission Regulation (EC) N°
2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs allows
the presence of 100 CFU/g in some ready-to-eat products. Conventional bacteriological
methods for the detection and quantification of L. monocytogenes are laborious
and time-consuming. The purpose of this study was to develop a rapid and
automated method for detection and quantification of L. monocytogenes in meat.
The target of the RealTime PCR was a portion of hly gene and the procedure consists
of two automated steps: DNA extraction, and set up of a fast RealTime PCR
mix. The whole process could be completed within few hours minimizing the risk
of carry-over contamination. To evaluate the inhibitor effect of food matrix on the
PCR an internal amplification control was included in each reaction and coamplified
using the same primer pair used for the target amplification. The specificity of
the assay was confirmed on 3 L. monocytogenes strains and 26 other bacterial
strains. The method was able to detect 2 genomic equivalents (GE) in 33/42 replicates
(LOD= 4.8 GE/reaction with a probability of 95 %) and the efficiency resulted
between 90 % and 98 % calculated by analyzing 7 standard curves based on six 10fold
dilutions starting from a 10 7 CFU/ml broth culture of L. monocytogenes. The
quantification range was linear over 6 log units with a square regression coefficient
> 0.99. Application of the assay was assessed with 12 artificially contaminated
samples of canned meat and with 8 real samples (smoked salmon, fresh fish
and salami). The microbial load was tested in comparison with a pour plate counting
method. The RealTime PCR was able to define an accurate quantification up to
10 3 CFU/g both in naturally and in artificially contaminated samples.
Bibliographic study concerning procedures for preparing environmental
samples for analyses, regarding to L. monocytogenes
Barre, L., Carpentier, B. and Gnanou Besse, N.
AFSSA, France
Listeria monocytogenes (L. monocytogenes) is a bacterium that can contaminate
foods and cause a mild non-invasive illness or a severe, sometimes life-threatening,
illness. L. monocytogenes is widespread in the environment. It can be readily isolated
from humans, domestic animals, raw agricultural commodities, and food processing
environments. Sanitation controls include effective environmental monitoring
programs designed to identify and eliminate L. monocytogenes in and on
surfaces and areas in the plant. The conditions are likely to be present in food factories
that may give rise to the development of persistent L. monocytogenes strains.
This persistence can be observed in spite of the well applied procedures of hygiene.
This document is a bibliographic study concerning procedures for preparing
environmental samples for analyses, regarding to L. monocytogenes. Different procedures
for collecting environmental samples are described: several procedures
for sampling methods are compared (cotton swab, sterile sponge, composite-ply
tissues, sonication, direct contact agar…). We have also compared different detection
and enumeration methods. We concluded that swabbing is known not to detach
all micro. The removal efficiency depends on the swab material. Swabbing in
moistened condition, as processed here, is more efficient than dry swabbing. Use of
ultrasound is still considered as a good method for detaching microorganisms.
However, ultrasounds can be lethal to bacteria, particularly when used at low frequency
and are more detrimental to attached bacteria than to suspended ones. We
concluded that it is important to adapt the type of sampling tools and techniques
to the type of surfaces and sampling locations.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Persistent L. monocytogenes isolates from Austrian and Irish dairies
show the same phenotypic and genetic background
Stessl, B.
Veterinary University Vienna, Austria
The food borne pathogen Listeria monocytogenes continues to be of major epidemiological
interest to food industry and food sciences. The Zoonosis Report
2004 of EFSA (European Food Safety Authority) listed 1267 cases of listeriosis in
Europe in 2004, resultant 0.3 cases per 100 000 capita. Human infections with L.
monocytogenes are rare, but are important due to the high mortality rate. This
study is focused on the PFGE (Pulsed Field Gel Electrophoresis) and FTIR
(Fourier Transform Infrared Spectroscopy) typing of L. monocytogenes strains isolated
at distant sampling points (1996 – 2007) in the Austrian and Irish dairy chain.
The aim of this research was to analyse if genetically indistinguishable (“persistent“)
L. monocytogenes clones can be differentiated with respect to their phenotype
(adaptation towards sanitizers and hygienic measures). A set of L. monocytogenes
isolates from seven different European dairies was analysed with two
epidemiological techniques: PFGE and FTIR. The PFGE method was performed
according the CDC PulseNet Standardized Protocol for Molecular Subtyping of L.
monocytogenes. Further data analysis was completed by Fingerprinting II Cluster
Analysis using Dice coefficient and UPGMA logarithm (Biorad, Austria). The FTIR
sample preparation and spectra analysis were performed as described by Oberreuter
et al. For data processing the program OPUS FT-IR (Bruker, Germany) was
used. Cluster analysis was performed using Ward’s algorithm. The PFGE as well as
the FTIR cluster analysis suggests the ability of short and long-time persistence of
L. monocytogenes isolates in soft, semi-hard and hard cheese manufacturing. An
explanation could be that the tested persistent L. monocytogenes isolates were not
exposed to selection due to sanitation (colonization of ecological niches characterized
by a low hygienic pressure). Additional studies will concentrate on the testing
of a L. monocytogenes strain subset against their disinfectant susceptibility.
Epidemiological data on listeriosis in Portugal: 2003 – 2008
Almeida, G, Magalhães, R., Hogg, T. and Teixeira, P.
CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal
The invasive form of listeriosis although rare is a very serious disease with a high
mortality rate. Young, Old, Pregnant, Immuno-compromised, sometimes referred
as YOPI, are identified as at risk groups. However the infection may also occur in
people with no known predisposing factors. In Portugal listeriosis is a not a notifiable
disease and is likely to be under-reported. In 2003 an incidence of 1.4 cases per
million inhabitants was reported. As in other European countries the number of
cases has increased in the last years. The aim of this study is to present epidemiological
on human cases of listeriosis occurred in Portugal between 2003 and 2008.
It was possible to gather 107 cases: 16 % were maternal/neonatal infections and
84% were non-maternal/neonatal infections. Concerning non-maternal/neonatal
cases the sex ratio (M/F) was 1.73, the mean age of the patients was 62 years old
with 46 cases (51 %) in individuals aged 65 or more. The microorganism was mainly
isolated from blood (59 %), from CSF (32 %) and from other specimen (9 %). The incidence
of listeriosis in Portugal based on voluntary reporting is lower than the European
notification rate (0.3/100000 inhabitants) and similar to countries like Austria,
Estonia, Latvia, Slovakia, Slovenia and Spain. The need of an active
surveillance system of listeriosis was clearly demonstrated.
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AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Diversity among Listeria monocytogenes isolated from humans
Kalekar, S. 1*, Rodrigues, J. 1, D’Costa, D. 1, Malik, S. V. S. 2, Kalorey, D. R. 3, Chakraborty, T. 4
and Barbuddhe, S. B. 1
1. ICAR Research Complex for Goa, India
2. Indian Veterinary Research Institute, Índia
3. Nagpur Veterinary College, India
4. Institute of Medical Microbiology, Justus Liebig University, Germany
Listeria monocytogenes is a food borne pathogen associated with severe diseases in
humans and animals. It is associated with neural, visceral and reproductive clinical
entities, leading to septicaemia, abortions, stillbirth, meningitis and meningoencephalitis,
especially in individuals at risk. We present the genotypic analysis
of 17 L. monocytogenes isolates recovered from humans in India during 2006–2009
by multiplex serotyping PCR allowing serovar predictions, conventional serology
and by PFGE. Multiplex-PCR serotyping assay revealed 88.24 % (15/17) of the
strains belonging to serovar group 4b, 4d, 4e, 11.76 % (2/17) to serovar group 1/2b,
3b. Twelve (70.59 %) L. monocytogenes isolates were assigned to serotype 4b, 2
(11.76 %) serotype 4d, one (5.88 %) serotype 4e and 2 (11.76 %) to serotype 1/2b by
serology. Ten ApaI pulsotypes were recognized among the 17 human isolates.
PFGE analysis allowed discrimination among isolates of the same serotype and
among isolates from the same sampling areas or those isolated from different
areas. Thus, PFGE together with multiplex-PCR serotyping allows rapid discrimination
of L. monocytogenes strains. In addition, the predominance of L. monocytogenes
serotype 4b is of concern, as this serotype has been most frequently associated
with human listeriosis outbreaks.
* Participation Supported by IUFoST.
Characterization of Listeria isolated from seafood
Rodrigues, J.*, Kalekar, S., Bhosle, S. N., Doijad, S. and Barbuddhe, S. B.
ICAR Research Complex for Goa, India
Listeria monocytogenes, the causative organism of listeriosis, is primarily transmitted
to humans through contaminated food. While several reports indicate that
fish and fishery products can be frequently contaminated with L. monocytogenes,
no major outbreaks associated with these products have been reported. Since fish
and fishery products may be a vehicle for L. monocytogenes, it is important to have
information on the incidence of this pathogen. The presence of L. monocytogenes
has been documented in the tropical environment but the incidence in fish and
fish products is low. We examined the prevalence, molecular characteristics and
virulence potential of L. monocytogenes isolates from fishery products to gain further
insights on the public health risk caused by this important food borne
pathogen. A total of 221 raw seafood samples were examined for the presence of
Listeria species. Of these 37 (16.74 %) samples were positive for Listeria species.
Out of these, 4 (1.8 %) isolates were confirmed as L. monocytogenes. Other species
detected was L. innocua (33). The hlyA gene was detected in all the L. monocytogenes
isolates. All the L. monocytogenes exhibited phosphatidyl inositol specific
phospholipase C activity on ALOA agar. Multiplex PCR based serotyping revealed
three L. monocytogenes isolates to be 1/2a, 1/2c, 3a, 3c group. One isolate belonged
to 1/2b, 3b serogroup. The isolates were grouped into three AscI PFGE profiles.
Prevalence of L. monocytogenes in fresh seafood is of significance as it may contaminate
and persist in the processing environment.
* Participation Supported by IUFoST.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Characterization of Listeria species isolated from milk
D’Costa, D. 1*, Bhosle, S. N. 2, Dhuri, R. B. 3, Kalekar, S. 1, Rodrigues, J. 1, Doijad, S. P. 1
and Barbuddhe, S. B. 1
1. ICAR Research Complex for Goa, India
2. Department of Microbiology, Goa University, India
3. Goa State Cooperative Milk Producers’ Union Limited, Curti, Ponda, Goa, India
Listeria monocytogenes is a foodborne pathogen commonly found in food and environment.
This study aimed to determine the occurrence of L. monocytogenes in
raw and market milk. Seven hundred sixty seven raw and market milk samples
were collected at different levels of collection and processing. The samples taken
included cow milk collected at udder level, from the milk cans, milk receiving centres,
processing unit and market. Samples were enriched in Listeria enrichment
broth and incubated for 48 h, followed by plating on PALCAM agar. Presumptive L.
monocytogenes isolates were purified and confirmed by PCR targeting the hly gene.
Overall, 10.56 % of the samples (81 of 767) were positive for Listeria species. Out of
these 37 (4.82 %) were confirmed as L. monocytogenes. Other Listeria species isolated
were L. innocua (5.47 %), L. ivanovii (0.13 %) and L. grayi (0.13 %). Maximum
isolates were recovered from samples collected from market followed by samples
at milk processing unit. L. monocytogenes were serotyped using multiplex PCR
method. A larger proportion of isolates (26) belonged to group corresponding to
serovars 1/2a, 1/2c, 3a, and 3c. Serogroup corresponding to serovars 4b, 4d and 4e
was detected in two strains while serogroup 1/2b, 3b, 4b, 4d, and 4e was detected in
nine strains. . This study demonstrates the prevalence of L. monocytogenes in the
raw and market milk and the need for good hygiene practices to prevent its entry
into the food chain.
* Participation Supported by IUFoST.
Prevalence of Listeria monocytogenes in chicken production chain
in Thailand
Kanarat, S., Nijthavorn, N. and Sukhapesna, J.
Veterinary Public Helath Laboratory, Bureau of Quality Control of Livestock Products, Department
of Livestock Development Phaya-thai Rd., Bangkok, Thailand
This study was conducted from 2004 to 2009 to investigate the prevalence of Listeria
monocytogenes in the chicken production chain in Thailand, i.e., from primary
production stage to further processing plants. Samples were taken from 43
breeder farms, 32 hatcheries, 1331 broiler farms, 22 slaughterhouses and 22 further
processing plants. Various types of samples were collected: fecal drops on the litter,
drinking water, and chicken feed from breeder and broiler farms; swabs and
mecomium from hatcheries; cloacal swabs and fresh chicken meat from slaughterhouses;
and ready-to-eat chicken products from further processing plants. The
study showed that there was no L. monocytogenes contamination in the chicken
production chain except in slaughterhouses and further processing plants. This
implies that the chickens did not carry the organism into slaughterhouses, and
consequently primary production practices were not responsible for the the contamination
of end products. This suggested that the observed L. monocytogenes
contamination in 2.5 % and 0.2 % of samples of fresh chicken meat and ready-toeat
chicken products, respectively, was due to the breakdowns in the application of
good hygienic and/or good manufacturing practices.
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AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Listeria monocytogenes in Ireland – Epidemiology and Molecular Typing
Cormican, M. G. 1, DeLappe, N. 2, McKeown, P. 3 and Garvey, P. 3
1. NUIGalway, Ireland
2. National Salmonella Reference Laboratory Ireland, Ireland
3. Health Protection Surveillance Centre, Ireland
Since January 2004 listeriosis is a notifiable disease in Ireland. Clinicians and laboratory
directors must inform the Medical Officer of Health of all cases diagnosed.
Information is collated by the national Health Protection Surveillance Centre.
Isolates of Listeria spp. from human cases and from food products may be voluntarily
submitted to the National Salmonella Reference Laboratory. For the years
2004 to 2007 11, 12, 7 and 21 (total =51) cases of listeriosis were reported to the
HPSC. There were 37 cases categorized as adult or juvenile, 10 pregnancy-related
and 4 neonatal. Cases were distributed through out Ireland with a slight seasonal
peak in Summer in most years. For the years 2004 to 2008 the NSRL has received
4, 4, 1, 12 and 14 (total of 35) L. monocytogenes isolates from humans and 6, 34, 16,
18 and 12 (total of 86) from foods. Isolates from human cases are predominantly
serotype 4b (n = 24). Isolates from food are predominantly serotype 1/2 (n= 74).
Pulsed field gel electrophoresis (PFGE) performed by the Pulse Net method with
ApaI and AscI enzymes and analysed with Bionumerics software ahs allowed the
definition of 10 clusters of closely related isolates (2 to 4 isolates per cluster). The
clusters link isolates from apparently sporadic human cases, including cases occurring
in different years and link isolates from food items with human cases. As
the clusters are small and it is not possible to perform typing in real-time epidemiological
links between cases have not been identified. The number of cases of
listeriosis in Ireland has increased in recent years. There is laboratory evidence
linking individual cases and linking cases with food. Real time molecular typing of
isolates is essential to identify linked cases in time frame that would allow timely
epidemiological investigation and intervention.
Characterization of Listeria monocytogenes isolated from human cases
of listeriosis occurred in Portugal in 2008
Magalhães, R.*, Barbosa, J., Santos, I., Almeida, G. and Teixeira, P.
CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal
Listeria monocytogenes is a Gram-positive foodborne pathogen that can cause serious
infections in humans. High-risk groups include the elderly, immunocompromised
individuals, pregnant women and their neonates Clinical manifestations
of invasive human listeriosis include meningitis, encephalitis, late-term spontaneous
abortion, and septicaemia. The fatality rate is high: 20 to 30 %. Foodborne
transmission of L. monocytogenes can also cause a self-limiting acute febrile gastroenteritis
in immunocompetent persons. Listeriosis is an infection of considerable
public health concern. In 2008, 27 isolates of Listeria monocytogenes were recovered
from Portuguese human cases of listeriosis and have been characterized
by biotyping (cadmium and arsenic sensitivity), genoserotyping and typing by
pulsed field gel electrophoresis (PFGE) using the enzymes AscI and ApaI. Strains
were aggregated in three multiplex PCR serogroups: IVb (66.7 %), IIb (25.9 %) and
IIa (7.4 %). Concerning biotyping four groups were found: sensitive to arsenic and
cadmium (51.9 %), arsenic sensitive and cadmium resistant (14.8 %), resistant to arsenic
and sensitive to cadmium (29.6 %) and resistant to both heavy metals (3.7 %).
Twenty two pulsotypes were indentified. The more prevalent pulsotype aggregates
three isolates, of these, two were time and geographical related. Two other pulsotypes
aggregate two time and geographical related isolates. It was shown that seven
pulsotypes (32 %) were also found in previous years.
* Participation Supported by IUFoST.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Post-processing environmental contamination of surface-ripened
soft cheese during affinage
D’Amico, D. and Donnelly, C.
University of Vermont, USA
Environmental sampling conducted in a cheese aging room identified extensive
contamination with Listeria monocytogenes. Further investigation revealed cross
contamination of washed rind cheeses aging on various racks. To determine the
extent of post-processing contamination of cheeses in this facility, two commercially
available PCR assays were investigated as tools for detection of L. monocytogenes
in naturally contaminated surface-ripened soft cheeses. Methods were
culture confirmed on CHROMagar Listeria. For analysis, cheeses were composited
and 10g sub-samples were assigned to an enrichment broth or Butterfield’s
Phosphate buffer for enumeration. Cheeses identified as positive for L. monocytogenes
by PCR and/or culture were subsequently enumerated. To achieve a detection
limit of ≥ 5 cfu/g, composited samples were diluted 1:5 followed by direct plating
of 1 ml on CHROMagar Listeria. Contamination levels ranged from < 5 to
1,600,000 cfu/g and varied by cheese type with the highest contamination levels
(mean ~356,000 cfu/g) observed in the smallest cheese variety (~200 g) and lower
levels (mean ~1,500-2,500 cfu/g) in the larger cheese types (~400-2000 g). Preliminary
ribotyping of random isolates recovered from each of the cheese varieties
identified a single lineage II ribotype, DUP-1030B. Based on PCR and cultural
results, 10g samples of cheese contaminated at levels as low as < 5 cfu/g were
identified following enrichments of 24 or 26 hours or after secondary enrichment
for a total of 44-48 hours. Single enrichment procedures of 24 and 26 hours in
Buffered Listeria Enrichment Broth and Oxoid 24LEB, respectively, followed by
plating 100µl on CHROMagar Listeria identified approximately 90 % of the true
positive samples. Secondary enrichment in MOPS-BLEB increased the sensitivity
to nearly 97 %. PCR detection following single primary enrichment was less sensitive
when compared to cultural techniques whereas dual enrichment PCR methods
were comparable to their cultural counterparts.
Genetic diversity of Listeria monocytogenes isolated
from Portuguese cheeses
Almeida, G., Magalhães, R., Santos, I., Barbosa, J., Hogg, T. and Teixeira, P.
CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal
The invasive form of listeriosis is a rare but serious disease with a high mortality
rate. Cheese has been associated with several outbreaks of human listeriosis involving
large numbers of consumers, with a wide economic and public health impact.
To reduce the incidence of listeriosis it would be necessary to reduce the
contamination level at production and at retail, or to inhibit the growth of Listeria
monocytogenes in high-risk foods. Typing of bacterial isolates reveals the relationship
among isolates. They can be used to determine the primary sources of bacterial
contamination and to link people who are ill following the consumption of
contaminated foods to the sources of bacterial contamination. Eighty isolates of L.
monocytogenes recovered from cheeses (40 from cheeses purchased at retail, 23
from cheeses ready to go to the market, 16 from cheeses analysed at Autoridade de
Segurança Alimenter e Económica laboratory and one isolate from cheese in quality
control routine activities) were typed by multiplex PCR serogrouping and by
PFGE using AscI and ApaI restriction enzymes.
Isolates were distributed by four MPCR groups: IIa (11 %), IIb (30 %), IIc (11 %)
and IVb (48 %). Strains involved in human episodes in Portugal were mainly IVb
(72 %) followed by IIb (18 %) and IIa (10 %). The combination of the profiles obtained
with both restriction enzymes (AscI and ApaI) yielded a total of 26 pulsotypes.
Eight of these pulsotypes were previously isolated from clinical cases of listeriosis
in Portugal. This study highlights the possibility of cheeses being
responsible for human cases of listeriosis in the country.
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Prevalence of Listeria spp. in retail raw ground beef in Izmir, Turkey:
A comparison of standard cultural method and Fluorescent
in situ Hybridization (FISH) technique for detection
Handan Baysal, A.*
Izmir Institute of Technology, Department of Food Engineering, Izmir, Turkey
Fluorescent in situ hybridization (FISH) is based on the principle that hybridization
of a nucleic acid sequence target of a microorganism with a specific DNA
probe labeled with a fluorochrome and imaging by a fluorescence microscope.
FISH method using a fluorescent labelled oligonucleotide probe designed from
specific DNA and RNA sequences has proven to be a useful tool for the detection of
a specific microorganism in environmental and clinical samples however the application
of the method in food microbiology is investigated by the research studies.
It usually involves four steps: sample fixation and permeabilization; hybridization
of the target sequence and the fluorescent probe; stringency washes;
and detection of the hybridized cells. In this study the possibility to use FISH
method for detecting a food-borne pathogen Listeria spp. that used as food safety
index in ground meat in market place of Izmir was evaluated. The work consists of
two steps. In the first step specified pathogen bacterium was inoculated in ground
beef and the applicability of FISH method was evaluated and in the second step the
pathogen was detected in fresh ground beef by standard (conventional) cultural
methods and FISH method. High correlation (r = 0.96) was found between FISH
and the conventional plate count method. The FISH protocol revealed to be specific
for Listeria spp. detection, since all bacteria showed a positive hybridization
signal. Results obtained from the direct application of the FISH technique to cultures
show that it allows the detection of Listeria spp. in hours.
* Participation Supported by IUFoST.
Using ListexP100 for Listeria monocytogenes detection in foods
Flores Lopes, J. 1, Ferreira Leite, I. 1, Azeredo, J. 2, Gibbs, P. 1 and Teixeira, P. 1
1. CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal
2. IBB / Institute for Biotechnology and Bioengineering, Centre of Biological, Engineering,
Universidade do Minho, Campus de Gualtar, Braga, Portugal
Food types susceptible of being contaminated with Listeria monocytogenes, such as
cheese, smoked fish, salads, raw vegetables, sausages and, in general, ready-to-eat
foods, pose a serious threat to consumers as this pathogen can cause severe diseases.
L. monocytogenes can multiply even at refrigeration temperatures, with the minimum
infective dose still remaining unknown. Development of efficient and rapid methods
for the a priori detection of contamination by this microorganism in foods is of great
significance and is required in order to ensure public health concerning consumption
of foods that are considered to be of higher contamination risk. The vast majority
of Listeria-specific detection methods, developed as alternatives to the standard
selective plating methods, possess drawbacks such as a low specificity or efficiency
(which is the case of antibody-based assays), or also detect dead cells (in the case of assays
that detect genetic material, e.g. PCR, restriction-enzyme pattern-based assays).
The main goal of the research work described herein was to investigate the possibility
of using ListexP100 (a commercial bacteriophage-based preparation for the control
of Listeria contamination in foods, gently supplied by EBI Food Safety B.V.) for the
design of a fast-response detection method for L. monocytogenes in foods. The procedure
developed during our research effort encompassed a bacteriophage amplification
assay based on the specificity and lytic activity of the broad-host range P100
listeriaphage with plaque formation as the assay end point. Aliquots of phage P100
were added to the test samples allowing all Listeria cells to be infected. After 15 minutes
of incubation at 30ºC, to allow phage adsorption and eclipse phase with the nucleic
acid delivered into the host cell, a virucidal agent was added in order to destroy
all those phages that have not effectively infected a bacterial cell. Among several effective
virucidal agents we selected 4.8 mM FeSO4 and 13 ppm of tannic acid and left
standing for 5 min at room temperature. The virucidal activity was neutralized by the
addition of 2% Tween-80 and the samples were then mixed with helper cells and
spread over the surface of agar plates using a soft agar overlay. After 16h of incubation
at 30ºC, the levels of Listeria contamination were determined by the presence of
phage plaques. It was demonstrated that the method was able to detect the presence
of bacteria, however only in samples containing high numbers of L. monocytogenes.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Mapping of molecular profiles associated to Listeria monocytogenes
food isolates circulating in Italy
De Cesare, A. 1, Parisi, A. 2, Latorre, L. 2 and Manfreda, G. 1
1. Alma Mater Studiorum – University of Bologna, Italy
2. Istituto Zooprofilattico Sperimentale della Puglia e Basilicata, Italy
Listeria monocytogenes is a bacterial pathogen transmitted to humans from a variety
of foods. Although most human listeriosis infections in Italy are being generally
considered to represent sporadic cases, recent results suggest that a considerable
number of listeriosis might occur in clusters, many of which could
represent single-source outbreaks. To support tracing of food sources of human
listeriosis infections, a long term mapping of serotypes, ribotypes and AFLP types
associated to Listeria monocytogens isolates collected from different meat and
cheese products as well as food production plant environments has been performed.
The results achieved show that AFLP profiles of Listeria monocytogens
isolates from the same food source cluster together with a similarity ≥ 84 %,
whereas AFLP clusters grouping isolates from different sources show a similarity
ranging between 60 and 78 %. Isolates belonging to the same AFLP cluster show
common ribotypes and serotypes. In particular, isolates from meat based products,
such as fresh and fermented sausages, are mainly classified within EcoRI ribotypes
14 S2 and 202 S2 and serotypes 1/2c and 1/2b. Furthermore, cheese products
are classified within the EcoRI ribotype 204 S5 and serotype 1/2a.
Zoonotic aspects of Listeria monocytogenes isolates
from zebu dairy animals
Parihar, V. S. 1,3, Barbuddhe, S. B. 1, Kalorey, D. R. 2, Kotwal, S. 4,Danielsson Tham, M.-L. 3
and Tham, W. 3
1. ICAR Research Complex for Goa, Ela, Old Goa, India
2. Department of Microbiology, Maharashtra Animal and Fishery Sciences University, Nagpur, India
3. Örebro University, Sweden
4. Sher-e- Kashmir University of Agricultural Sciences & Technology of Jammu, J&K
Listeria monocytogenes is a non acid-fast, Gram-positive pathogen, which is considered
food- and feed-borne. Knowledge of the direct and indirect transmission of
L. monocytogenes between animals and humans, is however, limited. The aim of
the current study was to characterize isolates of L. monocytogenes obtained from
zebu dairy animals with mastitis. Fifteen isolates of L. monocytogenes from the
same number of clinical and subclinical cases of mastitis were obtained from five
farms harbouring altogether 90 zebu dairy animals. The isolates were characterized
by serotyping, multiplex PCR (plcA, hlyA, actA and iap genes) and pulsedfield
gel electrophoresis (PFGE) using Asc Ι and Apa Ι enzymes. All the isolates
characterized belonged to serovar 4b and exhibited four virulence associated genes
of L. monocytogenes. PFGE revealed that all isolates belonged to the the same
pulsovar. One of the reasons that all isolates belonged to the same pulsovar despite
being from different farms may be that certain types of L. monocytogenes are
adapted to specific niches. The presence of an identical pulsovar could also indicate
a common source of infection for the different farms. All the farms delivered
milk to the same dairy. The same pulsovar of L. monocytogenes was isolated from
zebu dairy animals with mastitis from five different farms.
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AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Performance of ALOA and Palcam agars for detection of Listeria
monocytogenes in naturally contaminated raw meat products
Gravato Rowlands, R. E. 1, Asturiano Ristori, C., Geraldes Martins, C. 1,
Jakabi, M. 1 and Gombossy de Melo Franco, B. D. 2
1. IAL, Brazil
2. USP, Brazil
The correct isolation and identification of Listeria monocytogenes in foods heavily
contaminated with competing microorganisms may be difficult, especially when
L. monocytogenes is present in low numbers. ALOA (Agar Listeria Ottaviani &
Agosti) is a selective chromogenic agar that minimises the growth of contaminating
organisms, and Listeria spp. grow on this medium producing blue/green
colonies, with pathogenic species producing a characteristic opaque halo after 24
h at 37 °C. Non-Listeria spp. produce white colonies. The aim of this study was to
evaluate the performance of ALOA for investigation of L. monocytogenes in 552
samples of naturally contaminated raw meat products, comparing the results to
those obtained using the conventional Palcam agar. Meat samples were collected in
supermarkets in the city of Sao Paulo, SP, Brazil, and comprised 138 beef sausages,
138 pork sausages, 138 ground beef samples and 138 chicken leg samples. Using
the ISO 11290-1:1996/Amd.1:2004 detection method, L. monocytogenes was present
in 234 (42.4 %) samples. ALOA was more effective than Palcam agar for isolation of
L. monocytogenes (p ≤ 0.05): 82 samples (35 %) were positive in ALOA only, 23
(9.8 %) in Palcam agar only, and 129 (55.1 %) samples were positive in the two
media simultaneously. When the ISO 11290-2:1998 enumeration method was used
95 samples (17.2 %) were positive for L. monocytogenes, and among them, 31 samples
(32.6 %) were positive in ALOA only, 21 (22.1 %) in Palcam agar only, and 43
(45.3 %) samples were positive in the two media simultaneously. However, counts
of L. monocytogenes using the two media did not differ significantly (p > 0.05). Results
of this survey indicate that the use of ALOA both for detection and enumeration
of L. monocytogenes increases the effectiveness of the two ISO methods when
raw meat products are tested.
Comparison of MOPS-BLEB and Fraser as secondary enrichment
broths for Listeria monocytogenes
Upham, J., Huszczynski, G., Mosher, M., Borza, A., Dorey, M., Bosley, J., Hara, K.,
Mutanda, C., Liu, J., Byrne, B. and Douey, D.
Canadian Food Inspection Agency
The two methods used by CFIA laboratories for detection of L.monocytogenes from
foods employ bipartite selective enrichment procedures to increase pathogen concentration
prior to detection. The methods use the same bacteriological media for
primary enrichment but different secondary enrichment media. Secondary media
MOPS-Buffered Listeria Enrichment Broth (MOPS-BLEB) and Fraser broth were
compared for their ability to facilitate growth of L. monocytogenes. Foods contaminated
with Listeria were subjected to a common primary enrichment followed by
parallel secondary enrichment in MOPS-BLEB and Fraser broth. Listeria was isolated
and enumerated by plating the broths on selective agar. Of 164 food samples,
presumptive L.monocytogenes colonies were isolated from 164 using MOPS-BLEB
and 162 using Fraser broth, demonstrating broth equivalence (p = 0.50, Chisquare).
Quantitative analysis revealed significantly higher levels of L.monocytogenes
in MOPS-BLEB than Fraser broth (log10 CFU, 8.88 and 7.99, p < 0.0001 by
paired t test). Interestingly, for foods inoculated with a mixture of L.monocytogenes
and a non-pathogenic species of Listeria, L.innocua, concentrations of the
later were not significantly different in MOPS-BLEB compared to Fraser broth
(log10 CFU, 8.85 and 8.47, p = 0.06), resulting in a higher ratio of L.monocytogenes
to L.innocua in MOPS-BLEB. Enrichment broths used by CFIA were found to be
equivalent for enabling cultural detection of L. monocytogenes, however, MOPS-
BLEB produced significantly higher pathogen concentrations and pathogen to
competitor ratios and thus has potential to enable earlier detection and facilitate
detection when competitors are present.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Comparison of UVM, Palcam and Oxoid Novel Enrichment (ONE) broth
as primary enrichment broths for Listeria monocytogenes
Upham, J., Mosher, M., Borza, A., Huszczynski, G., Dorey, M., Eloranta, K. and Douey, D.
Canadian Food Inspection Agency
The purpose of this study was to compare three primary enrichment broths for
ability to facilitate detection of Listeria monocytogenes in foods: 1) UVM I, 2) Palcam,
and 3) a single-step enrichment broth from Oxoid Company, ONE broth.
Media was directly inoculated with healthy or heat-stressed L. monocytogenes and
incubated for up to 48 hr, and growth curves were constructed by diluting and plating
the enrichment cultures at regular time intervals to determine cell concentration
(cfu/ml). Growth curves were also prepared from media that had been used to
enrich various food products (deli meats, dairy products, fresh produce and
seafood) that were artificially contaminated with L. monocytogenes alone, or L.
monocytogenes in the presence of the background organisms Escherichia coli and
Staphylococcus epidermidis, or in the presence of a non-pathogenic Listeria species,
L. innocua. In the absence of food matrices or competitive microorganisms, the lag
phase of heat-stressed L. monocytogenes was significantly shorter in Palcam broth
(6.5h) compared to UVM and ONE broth (each 7.8h), and the exponential growth
rate was significantly greater in Palcam broth (0.371) and ONE broth (0.396) compared
to UVM broth (0.277). Similar differences in growth rates were observed
when broths were used to enrich L. monocytogenes-contaminated food products.
When food were contaminated with a equal mixture of L. monocytogenes and L. innocua,
the exponential growth rate of L. innocua was notably faster than L. monocytogenes
in UVM and Palcam broth, whereas the opposite was true in ONE broth.
In addition, the presence of L. innocua caused a decrease in L. monocytogenes
growth rate in Palcam broth and to a lesser extent in UVM broth, but not in ONE
broth. These studies suggest that ONE broth may be preferable to UVM and Palcam
broths to facilitate detection of L. monocytogenes when L. innocua is present.
Isolation and characterization of Listeria monocytogenes from asazuke
(Japanese light pickles)
Maklon, K., Kusumoto, A., Makino, S.-I. and Kawamoto, K.
Obihiro Univ. Agri. Vet. Med., Japan
Asazuke is a ready-to-eat Japanese light pickle, mainly made of vegetables which
are known to be one of the sources of Listeria monocytogenes contamination. Although
asazuke is a popular side dish in Japan, the hazard of bacterial contamination
has not been evaluated yet. In this study, we investigated the prevalence of L.
monocytogenes, Salmonella spp., Escherichia coli O157 and coliforms in 108 asazuke
samples that randomly collected from supermarkets in Obihiro (Hokkaido prefecture,
Japan) during the period of June to November 2007. Twelve (11.1 %) L.
monocytogenes were isolated with predominant serotype 4b (7 isolates) followed by
1/2a (2 isolates), 1/2b, 3b and 4c (1 isolate each) while Salmonella spp., E. coli O157
and coliforms were not detected. All L. monocytogenes isolates demonstrated hemolytic
activity by CAMP test and possessed all the virulence-associated genes
(prfA, actA, mpl, inlA, inlC, plcA, plcB, hly, iap, clpC and opuCA) by PCR, those revealed
their potential pathogenicity. Moreover, 7 out of 12 isolates were from
asazuke produced by one factory and pulsed-field gel electrophoresis (PFGE) profiles
suggested that 6 of them were indistinguishable. Additional investigation of L.
monocytogenes contamination was performed in the asazuke factory environment
and 23 out of 60 swabs (38.33 %) were isolated. Comparison of PFGE profiles
showed relatedness between food and environmental isolates. Taken together, our
results indicated that asazuke was contaminated with L. monocytogenes, which
was probably from vegetables, human sources or from production lines. Further
analysis to clarify the source of pathogen contamination is needed to establish a
prevention method and consumers should be aware of asazuke consumption that
might be contaminated with L. monocytogenes.
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AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Grouping of human Listeria monocytogenes isolates
Lopez-Valladares, G., Danielsson-Tham, M.-L. and Tham, W.
School of Hospitality, Culinary Arts & Meal Sciences, Örebro University, Grythyttan, Sweden
More than 800 clinical isolates of Listeria monocytogenes from humans in Sweden
were characterized by serotyping and pulsed-field gel electrophoresis. The restriction
enzyme used was AscI. The serovar 4b isolates were divided into 27 PFGE
types, the serovar 1/2a and 1/2c isolates into 108, and the serovar 1/2b and 3b isolates
into 24. PFGE types with identical profiles of small bands (< 145 kbp) were
grouped together with PFGE designations of A-Z. This grouping was based on the
assumption DNA rearrangements involving insertions, deletions, or transpositions
of genes are less common in the small fragments than in large fragments.
The 27 serovar 4b PFGE types were further divided into 5 groups and the 108
serovar 1/2a and 1/2c PFGE types into 16 groups. The serovar 1/2b and 3b types
(n = 24) were divided into five groups. Part of the collection of L. monocytogenes
isolates was analyzed with multiple-locus variable-number tandem-repeat
analysis and the clusters were generally in good agreement with both the clusters
generated by PFGE and serotyping data. The advantage of dividing L. monocytogenes
PFGE types into groups is that the profile of every new isolate characterised
with PFGE can easily and quickly be identified by studying the group affiliation of
the isolate and then identifying the matching type within the group.
Characterization of Listeria monocytogenes strains isolated from food
and environmental samples
Nucera, D. M. 1, Lomonaco, S. 1, Manila Bianchi, D. 2, Decastelli, L. 2, Grassi, M. A. 1 and Civera, T. 1
1. Università degli Studi di Torino, Italy
2. Istituto Zooprofilattico del Piemonte Liguria e Valle d’Aosta, Italy
This study aimed to investigate type diversity and distribution over time and
across sources by subtyping via Pulsed Field Gel Electrophoresis (PFGE) a set of
300 L. monocytogenes strains collected over a five year period. Five clinical human
strains isolated in the same geographic area and period of the study were also included
in the analysis. The isolated strains belonged to serotype 1/2a (45 % of the
samples), 1/2c (22 %), 4b/4e (16 %); however, 5 % of the strains were untypeable.
Significant associations were observed between serotype 1/2a with dairy (O.R.=
13.9; χ 2 p < 0.05) and 1/2c with meat (O.R.= 33.3; χ 2 p < 0.05). PFGE results highlighted
152 pulsotypes shared among two or more samples: 9 pulsotypes were recurrent
and 6 were shared between strains isolated from different food and environmental
sources. The other generated pulsotypes were source specific and/or
retrieved in one year only. These findings may indicate the presence of both nicheadapted
as well as ubiquitous PFGE types. Moreover, no PFGE types were shared
between strains collected from food/environmental isolates and human clinical
strains. The results of this research underline and confirm the importance of implementing
a broad typing database which could facilitate epidemiological investigations
and enhance novel food attribution modelling approaches for the identification
of listeriosis outbreaks and the related sources.These data, even if focused
on strains collected in a limited geographic area, may pose the ground to evidence
how large subtype databases may aid in the detection of common- and source specific-
types. However, more comprehensive databases, both at national and at European
level, are needed to reveal L. monocytogenes strains diversity and to provide
useful data for epidemiological investigations.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Development of a multiplex SNP-typing method to identify the four
epidemic clones of Listeria monocytogenes
Lomonaco, S. 1, Civera, T. 1, Dalmasso, A. 1, Knabel, S. J. 2 and Bottero, M. T. 1
1. Università degli Studi di Torino, Italy
2. The Pennsylvania State University, USA
The concept of epidemic clone (EC) is used to describe closely related strains of L.
monocytogenes belonging to geographically and temporally distinct outbreaks. Currently,
four ECs have been identified: ECI, ECII, ECIII, and ECIV (also ECIa). The
current classification of ECs is based on results from different subtyping techniques
such as RFLP analysis, hybridization assays, PFGE and ribotyping. Recently, a multiplex
PCR able to identify ECI, ECII and ECIII was developed. In addition, sequence-based
methods, such as multivirulence-locus sequence typing, were able
to further subtype EC strains and also to highlight potentially informative single
nucleotide polymorphisms (SNPs). The aim of the present study was to develop an
alternative method for the identification of all four ECs by developing a SNP-typing
method based on minisequencing. This technique simultaneously analyses several
SNPs based on a Primer-Extension Reaction (PER). Extension primers are designed
immediately adjacent to the SNPs and extended by a single [F]ddNTP. Results are
then visualized with a genetic analyzer as specific patterns. Six SNPs, yielding a
specific pattern for each EC, were selected. These SNPs were located on 4 virulence
genes (inlA, inlJ, inlB and srtA). Gene fragments were preliminary amplified with a
multiplex PCR and then used as template for the PER reaction. Analyzed samples
were 39 ECs strains, 25 strains from the ILSI Diversity Subset and 22 non ECstrains.
All ECs gave a clone-specific pattern. Moreover, 9 strains not previously
classified as ECs showed the same profile as ECI, ECII and ECIV. These findings
were confirmed with other biomolecular tests (e.g. ECs-specific PCR) and by considering
available epidemiological data for these strains. The proposed SNP-typing
assay is potentially high-throughput and amenable to automation, allowing a
rapid identification of all four ECs and can therefore be used as a screening method
in the analysis of L. monocytogenes strains.
Listeria monocytogenes incidents reported to the UK Food Standards
Agency from 2000 to 2008
Aish, J.
Food Standards Agency, UK
The UK Food Standards Agency contributes to the management of a wide range of
food related incidents involving microbiological, chemical, radiological and physical
hazards. One important foodborne microbiological hazard for which the
Agency oversees incident investigations is Listeria monocytogenes. When incidents
are reported to the Agency the relevant data is recorded on the Agency’s Incidents
Database. Notification is received from a wide range of food businesses, Local Authorities,
the Health Protection Agency and other Government Departments. Information
held on the database was analysed to determine the number of incidents
involving Listeria spp. that were reported to the Agency between 2000 and
2008 and to identify that main food categories that were involved. During the nine
year period from 2000 to 2008 approximately 140 incidents caused by the contamination
of food with Listeria spp. were reported to the Agency, the majority of
which involved L. monocytogenes. Most of the incidents involved ready-to-eat
meat/meat products, cheese, fish/shellfish and sandwiches/sandwich fillings. L.
monocytogenes is an important foodborne pathogen. Although healthy people are
not usually at risk of illness, listeriosis can be an issue for vulnerable groups such
as pregnant women and the immunocompromised. In addition to overseeing incident
investigations the Agency’s 4C’s strategy (cleaning, cooking, chilling and
avoiding cross-contamination) is important in reducing the risk from L. monocytogenes.
The Agency also provides food safety advice to vulnerable groups and
commissions research to further our understanding of this organism.
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AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Genetic diversity of Listeria monocytogenes in broiler flocks
Courtillon, C. Toquin, M.-T., Le Nôtre, Y., Fravalo, P. and Mansour Chemaly, M.
AFSSA, France
Listeria monocytogenes is a foodborne pathogen, persistent in industrial environment
and is responsible of listeriosis, an infectious and rare disease. The aim of
this study was to update and create data set from broiler flocks regarding their
contamination by Listeria monocytogenes. 145 flocks have been sampled, 85 conventional
and 60 free range flocks. Molecular characterization by PFGE with two
restriction enzymes ApaI and AscI was performed in order to establish the clonal
relationships of the isolates and to trace the contamination between the flocks. A
total of 126 isolates were genotyped. The prevalence in broilers has been estimated
at 31.7 % (46 positive samples on 145). The serotyping revealed the presence of
serotype 1/2a in majority: 50 % vs. 31.7 % for serogroup 4 (4e and 4b) and 18.3 % for
1/2b. Serotype 1/2a is the most present in conventional farms (37.3 %) contrary to
free range farms (12.7%) where serogroup 4 is dominant (23 % vs. 8.7 %). PFGE
typing showed a high discriminatory index: 0.971 for both combined enzymes. 47
genetic patterns were identified. Dendrograms, obtained with BioNumerics software,
confirmed the high genetic variability between isolates (55.7 % of similarity)
especially in serotype 1/2a (65.5 %). This index (0.954) was similar to the one
obtained when studying laying hen flocks. The serogroup 4 and the serotype 1/2b
seemed more clonal, 0.892 and 0.798, respectively. Regarding the production system,
results between conventional and free range farms are comparable, 0.959 and
0.948, respectively. The PFGE showed that two identical profiles can be found in
different flocks and different geographical locations while in a same flock, patterns
can be very different. The breeding type is not linked to bacteria genetic
identity as are serotypes. In conclusion, this investigation brought new and updated
data regarding Listeria monocytogenes in broiler flocks which were not available
before.
Tracing Listeria monocytogenes contaminations throughout
the processing chain of a typical Italian pork meat product
using Pulsed Field Gel Electrophoresis (PFGE) characterization
Annunziata Prencipe, V., Acciari, V., Torresi, M., Migliorati, G.,
Marfoglia, C. and Valentina Rizzi, V.
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy
Pulsed Field Gel Electrophoresis (PFGE) and serotyping analyses were performed
on 124 Listeria monocytogenes strains isolated from a prevalence study throughout
the Parma dry-cured ham production, in order to evaluate contamination patterns.
Strains had been isolated at three different points of the production process:
I) fecal samples/carcasses in slaughterhouses; II) fresh hams after cutting; III)
de-boned, dry-cured and packaged hams at the end of the production chain. Traceability
to corresponding feces, fresh hams and dry-cured hams had been assured
for all carcasses, so every strain was tested to evaluate the presence of contaminations
in previous or next phases of the production process. Genetic relationships of
56 different pulsotypes obtained from the combination of AscI and ApaI macrorestriction
patterns were graphically represented and analyzed. Six different clusters
were identified. The analysis of PFGE patterns obtained from the same sample
examined at the three different points of the production process (carcass – fresh
ham – cured ham) did not produce identical or highly similar pulsotypes. The only
pulsotype occurring in a strain from faeces was not recovered from other materials.
Strains from the same production plants were usually genetically homogeneous
or highly similar. In the plants where there was not genetic homogeneity,
one pulsotype or cluster was always sharply prevalent over the others. The presence
of single or prevalent pulsotypes in the same processing plants could be associated
to persistent strains of Listeria monocytogenes. This is corroborated by
the highest prevalence of 1/2c serotype strains, which adhere significantly more to
working surfaces, isolated from all stages of the process. Genetically homogeneous
or highly similar strains were also recovered in different establishments, at different
phases of the production process. This situation could be bounded to previous
sporadic contaminations between different production stages, which could
have allowed the localization of strains into favorable niches inside the processing
environment.

AREA // C // Epidemiologic and clinical aspects of Listeria monocytogenes and listeriosis
POSTER PRESENTATIONS // P / 82–125, 183–186
Production and validation of capture-ELISA kit based on monoclonal
antibodies specific for Listeria monocytogenes in foodstuffs
Portanti, O., Di Febo, T., Luciani, M., Pompilii, C., Armillotta, G., Principe, V.,
Lelli, R. and Semprini, P.
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy
A Listeria monocytogenes ELISA test was produced for use in the screening of
foods. Validation was carried out according to the criteria for qualitative analysis
set out in ISO 16140:2003. The ELISA kit has satisfied the norm requirements in
terms of relative accuracy, relative sensitivity, relative specificity, relative detection
level, inclusivity and exclusivity. The kit is a capture-ELISA test applied to
the sample enrichment broth, based both on a monoclonal antibody (9B8F7) to
L. monocytogenes and a peroxidase-conjugated anti-Listeria monoclonal antibody
(6F12C8). The method has been validated in meat, fish and dairy products by
means of incurred and artificially contaminated samples with target and non target
strains. Results for relative accuracy, relative sensitivity, relative specificity
and relative detection levels are in agreement with ISO 11290-1:1996 requirements.
Capture-ELISA test is able to detect Listeria with 100 % inclusivity and exclusivity
in the above mentioned matrices. Capture-ELISA kit was produced in a ready-touse
format and can be adopted by microbiological labs that need fast analytical
methods. It also shows the same features as the internationally recognised ISO
11290 techniques, as required by UNI EN ISO 17025:2005.
Production of a reference material for microbiological tests
containing Listeria innocua
Pomilio, F., Ricci, L, Di Giannatale, E., Semprini, P., Candeloro, L. and Migliorati, G.
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy
The study describes the production of a reference material constituted by glass
balls, covered by skimmed milk powder micronized and artificially contaminated
with Listeria innocua (ATCC 33090 strain). The lot of prepared material, constituted
by 1g unities of balls, has been made in test-tubes, submitted to a stability
and homogeneity evaluation through the determination of the mesophilic bacterial
count and following Listeria innocua identification. The material stability, kept
at –20 °C, has been evaluated at 54, 180 and 355 days. Data pointed out the decreasing
tendency of the bacterial count, statistically significant, at 54 days. At 180
and 355 days, the lot resulted stable. Regression analysis at 54 days (t-value -2,67)
has underlined a significant decreasing trend (p-value < 0.05). In the observed period
the decrease has been equal to 8.75 UFC/g. Samples examined in the same
time lapses resulted homogeneous in the interval between 180 and 355 days. Only
a sample out of the 105 examined resulted negative. After 355 days of preservation
at –20 °C, the challenge test has been carried out at 5 different temperatures
(-20 °C; +4 °C; +22 °C; +30 °C; +37 °C). The material evaluated with non-parametric
tests, for independent samples resulted stable at -20 °C and +4 °C. Furthermore, for
the reference value definition, a proficiency test has been organized, which allowed
to assign the value of 3.2 UFC/g to the material. In the same study, 35 samples
out of 244 (15.4 %) resulted negative. Used methodology could be applied to
reference materials production also on other micro-organisms. The adopted technology
has the benefit of operating in “closed” systems, making bio-safety of operators
and environment easily manageable.
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AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Colonization of a newly constructed commercial chicken further
processing plant with Listeria monocytogenes
Berrang, M. E. 1, Meinersmann, R. 1, Frank, J. 2 and Ladely, S. 1
1. USDA-Agricultural Research Service, USA
2. University of Geórgia, USA
This study was undertaken to determine potential sources of Listeria monocytogenes
in a newly constructed chicken further processing plant and document the
eventual colonization of the facility by this pathogen. To ascertain the colonization
status of the plant, floor drains were sampled after a production shift and again
after a clean up shift on a roughly monthly basis for 21 months. Potential sources of
L. monocytogenes to the plant included: incoming raw meat, incoming fresh air
and personnel. Nearby environment and community samples were also examined.
All L. monocytogenes detected were subjected to DNA sequence based subtyping.
L. monocytogenes was not detected in the plant before the commencement of processing
operations. Within four months, several subtypes of L. monocytogenes were
detected in floor drains both before and after cleaning and sanitizing operations.
No L. monocytogenes was detected on filters for incoming air, samples associated
with plant employees, or a nearby discount shopping center. One subtype of L.
monocytogenes was detected in a natural stream near the plant however, this subtype
was never detected inside the plant. Eight subtypes of L. monocytogenes were
detected in raw meat staged for further processing; one of the raw meat subtypes
was indistinguishable from a persistent drain subtype recovered after cleaning on
eight occasions in four different drains. Poultry further processing plants are likely
to become colonized with L. monocytogenes; raw product is an important source of
the organism to the plant.
Episcopic differential interference contrast/epifluorescence
microscopy to characterise in situ Listeria monocytogenes biofilms
on stainless steel surfaces
Gião, M. S. and Keevil, C. W.
University of Southampton, UK
Listeria monocytogenes is a Gram-positive pathogen associated with the contamination
of several food products, including meat, dairy products and vegetables. Although
this pathogen can be found in natural environments the contamination of
processed food can occur after contact with contaminated surfaces. Previous studies
routinely focused on biofilm recovery and culture quantification. In situ studies
utilised mostly SEM which is tedious, expensive and known to introduce artefacts.
By contrast episcopic differential interference contraste/epifluorescence
(EDIC/EF) microscopy is a novel and rapid light microscopy technique ideally
suited to study biofilms in a native state. The aim of this work was to utilise the advances
in EDIC/EF microscopy to study the formation of L. monocytogenes
biofilms under different nutrient and temperature conditions. L. monocytogenes
NCTC 13372 was suspended in Brain Heart Infusion (BHI), Tryptone Soya Broth
(TSB), TSB supplemented with 0.6 % of Yeast Extract (TSBYE) and filter-sterilised
tap water. Five ml of each suspension were transferred for 6 well plates containing
1 cm 2 stainless steel coupons and incubated at 4 °C, 22 °C, 30 °C and 37 °C. After 4
and 24 hours 2 coupons were removed for each tested medium. One coupon was
stained in situ with SYTO 9 and observed under EDIC/EF microscopy and the
other coupon was scraped and total and cultivable cells quantified by staining with
SYTO 9 and plating onto BHI agar, respectively. Results showed that even for the
lowest temperature, after 4 hours of incubation L. monocytogenes biofilms were
already developing on the surfaces. Except in the case of biofilms formed in tap
water where cells were in general less cultivable, the different media do not influence
significantly the formation of biofilm on stainless steel surfaces. Conversely,
the in situ observation of the coupons demonstrated that the morphology of the
biofilm is temperature dependent with implications for food processing.

AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Temperature dependent defect in biofilm formation
by Listeria monocytogenes
Abdalla, S. 1, Glenn, S. 1, Shama, G. 2 and Andrew, P. 1
1. Department of Infection, Immunity and Inflammation, University of Leicester LE1 9HN, United
Kingdom
2. Department of Chemical Engineering Loughborough University, Loughborough, LE11 3TU, United
Kingdom
Listeria monocytogenes can cause major problems in food processing industries because
of its ability to survive and grow over a wide range of environmental conditions.
Furthermore L. monocytogenes can attach to and produce biofilm on wide
variety of different surfaces in the food-processing environment, allowing the bacterium
to resist to antimicrobial and sanitizing agents. The aims of this study were
to investigate the ability of Listeria monocytogenes wide type strain 10403s and
Tn917-LTV3 transposon mutants to attach to abiotic surface at a variety of temperatures.
A published attachment assay was modified to compare the ability of the
WT strain and a number of transposon mutants to attach to polystyrene surfaces
over 2 hours incubation. Attached cells were detected using crystal violet staining.
Certain transposon mutants were shown to have a significant reduction in the level
of attachment, but this defect was temperature dependent, being evident at 30 °C
and 18 °C but not at 37 °C. The identity of the transposon insertion site in these
mutants is being actively pursued. It was concluded that there is a temperature dependent
involvement of some gene production in surface attachment.
Mode of action of Lactococcus lactis Sa31 antimicrobial peptide
on Listeria monocytogenes ½ c
Barile, M. 1, Mormile, A. 1, Ceres, C. 1, Pepe, O. 2, Cortesi, M. L. 1 and Murru, N. 1
1. DISCIZIA, Faculty of Veterinary Medicine, Napoli, Italy
2. Faculty of Agraria Università degli Studi di Napoli, Italy
Lactic acid bacteria produce antimicrobial peptides referred to as bacteriocins
that have potential as natural food preservatives. Lactococcus lactis Sa31, isolated
from gilthead breams fillets packaged in modified atmospheres, normally produces
bacteriocin in GM 17 broth with maximum activity (2.560 AU/ml) at 11 h in
the late logarithmic to early stationary phase of growth. In the present work was
investigated the Sa 31 ability of bacteriocin production in different liquid media,
the partial characterization and mode of action of peptide against Listeria monocytogenes
½ c strain. Overnight extracts of producer strain cultures in Triptone
Soya Broth, M 17 + 0.5 % glucose (GM 17 ) and 0,5 % lactose (LM 17 ), MRS, and BHI
broth was tested against Listeria monocytogenes ½ c. To evaluate the mode of action,
the crude (5.120 AU/ml -1) and partially purificated bacteriocin (10.240
AU/ml -1) by precipitation with ammonium sulfate were inoculated in suspension
of 50 mM potassium phosphato buffer pH 7.0 containing Listeria monocytogenes
½ c (1.4 x10 8 CFU/ml). Finally, for absorption study of partial purificated bacteriocin,
this was added at 10.240 AU/ml on cell of Listeria monocytogenes ½ c resuspended
in 100 mM NaCl pH 7.0 incubated for 30 min. at 20 °C. The residual
bacteriocin activity was tested as described above. The strain Sa 31 was able to
produce bacteriocin in BHI broth with maximum titre 5.120 AU/ml -1 after short
incubation time. The bacteriocin, sensitive to heat and more powerful at acid pH,
exhibited a bactericidal mode of action and acted rapidly. The death of Listeria
monocytogenes ½ c cells was observed within 10 min. after treatment with 10.240
AU/ml of bacteriocin (reduction in viable counts from 1.4 x 10 8 to 6.5x10 5 CFU/ml).
The bacteriocin was completely absorbed on the sensitive strain L. monocytogenes
½ c at pH 7.0. These results demonstrate the effectiveness of this bacteriocin and
its producer as an inhibitor of Listeria monocytogenes in vitro, and the possibility of
their applying in a food system where post-manufacture contamination by this
organism could be problematic as in fishery products as well as in other foods.
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AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Tracing the source, epidemiology and persistence of
Listeria monocytogenes in Irish Farmhouse cheese processing facilities
Jordan, K., Fox, E., O’Brien, M. and Hunt, K.
Teagasc, Ireland
Many outbreaks of listeriosis have been related to dairy products, although the
source of contamination has rarely been determined. Sixteen farmhouse cheesemaking
facilities were sampled at monthly intervals over a two-year period. Samples
included raw materials, food contact surfaces, non-food contact surfaces, farm
samples and product. In order to determine the source, persistence and epidemiology
of the strains obtained from 13 cheesemaking facilities, isolates of L. monocytogenes
were differentiated using PFGE and serotyping. At 9 of the 13 contaminated
processing facilities farm samples were the source of the environmental
contamination. We could not determine the source of the contamination at the
remaining 4 facilities. Persistent isolates (similar isolates obtained at the same facility
for > one year) were found at two cheesemaking facilities. Reduction of the
occurrence of L. monocytogenes in milk could reduce the contamination of cheese
processing facilities and therefore reduce the risk of cheese contamination. Cheese
producers can also take steps to reduce risk by reducing milk spillage at milk intake
and sterilising any area where milk was spilt. In addition, barriers between the
farm and processing facility will help reduce the risk of contamination.
Phenotypic and genotypic characteristics of Listeria monocytogenes
strains isolated from a convenience food-processing plant
Blatter, S., Stephan, R., Tasara, T. and Zweifel, C.
Institute for Food Safety and Hygiene, University of Zurich, Switzerland
L. monocytogenes as a food-borne pathogen has significant public health and economic
impacts. Amongst other foods, ready-to-eat products have been implicated as
routes for human infection. The aim of this study was to investigate the occurrence
and genetic diversity of L. monocytogenes isolated from the environment and food
products in a Swiss sandwich-producing plant. Over a 12-month period, environmental
swabs (n = 2.028) collected during the working activity and after cleaning
and disinfection, as well as ingredient and sandwich samples (n = 217) were collected.
Swabs originated from the processing room and the equipment of five sandwich
production lines. Isolation of L. monocytogenes was accomplished by culture
after enrichment (Half-Fraser/Fraser broth, Palcam and Ottaviani-Agosti agar).
Isolated strains were characterized by serotyping, determination of genetic lineages,
Rep PCR typing, and PFGE analysis after macrorestriction. L. monocytogenes
were detected in 70 (3.5 %) environmental swabs and 16 (7.4 %) samples from ingredients
and sandwiches. Of the positive environmental samples, 40 % originated
from three slicers, which tested repeatedly positive on several occasions. Of the 86
isolated L. monocytogenes strains, 93 % belonged to serotype 1/2a and genetic lineage
II, whereas the remaining strains were of serotype 1/2b and genetic lineage I.
Genotyping by Rep PCR and PFGE analysis yielded each six different profiles. Fiftyeight
(82.9 %) strains from the processing environment and 9 (56.3 %) from ingredients
and sandwiches belonged to only one genotype. Strains of this genotype were
found repeatedly on/in slicers, tables, conveyor belts, tables, a bread-feeding machine,
spattles, air guns, salmon and egg sandwiches. Based on the genotyping results,
indications of persistent contamination of the sandwich processing area with
a predominant strain were evident over the whole sampling period. Moreover, in
food-processing plants with low Listeria prevalence in the final product, environmental
monitoring is more effective than end product testing.

AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Influence of flow direction on the adhesion of Listeria monocytogenes to
brushed stainless steel surfaces
Skovager, A. 1, Whitehead, K. 2, Ingmer, H. 3, Verran, J. 2 and Arneborg, N. 1
1. Department of Food Science, Food Microbiology, Faculty of Life Sciences, University of Copenhagen,
Denmark
2. Division of Biology, School of Biology, Chemistry and Health Science, Faculty of Science and
Engineering, John Dalton Building, Manchester Metropolitan University, Manchester, England
3. Department of Veterinary Disease Biology, Section for Microbiology, Faculty of Life sciences,
University of Copenhagen, Denmark
The ability of Listeria monocytogenes to form biofilms on the surfaces of process
equipment constitutes a major health problem for the food industry. When the
bacteria have succeeded in forming a biofilm, they are difficult to remove from
the surface, due to their strong adhesion on the processing equipment. The innermost
cells of a biofilm are also protected against the action of cleaning- and disinfection
agents. It is therefore extremely important to target the bacteria before
they adhere to the solid surface and develop into a biofilm. One factor which may
influence the adhesion of L. monocytogenes is the surface characteristics of processing
equipment. In the present work, a method using fluorescence microscopy
and a perfusion system will be developed to examine the initial adhesion of single
L. monocytogenes cells to steel surfaces. Results will be presented showing the influence
of flow direction on the adhesion of L. monocytogenes to brushed stainless
steel surfaces.
Occurrence of Listeria monocytogenes in raw milk and dairy products
in Kazerun, Iran
Mehdi Mahmoodi, S. M. and Javanmardi, F.
Islamic Azad University – Kazerun Branch, Iran
The presence of Listeria monocytogenes was investigated in a total of 360 raw milk
and dairy product samples including white cheese, yoghurt and Iranian yoghurt
drink (Doogh) that were collected during five months from two traditional dairy
manufacturer in Southern Iran. The prevalence of Listeria monocytogenes in raw
milk and white cheese samples of manufacturer A was found to be 1.7 % and 3.3 %
respectively and of manufacturer B was found to be 3.3 % and 6.7 % respectively.
No Listeria monocytogenes was isolated in any of the yoghurt and Doogh samples of
both manufacturer probably because of their low pH values.
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AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Antilisterial mode of action of bacteriocin ST182Gu produced
by Enterococcus casseliflavus isolated from guava
Todorov, S. 1*, Destro, M. T. 1, Chiarini, E. B. 1, Vaz-Velho, M. 2 and Franco, B. D. G. M. 1
1. University of Sao Paulo , Brazil
2. ESTG, Viana do Castelo , Portugal
This research is on the anti-Listeria bacteriocin ST182Gu and studies its mode of action.
Bacteriocins of lactic acid bacteria (LAB) are ribosomally synthesized antimicrobial
peptides. Their bactericidal mechanisms may include pore formation, degradation
of cellular DNA, disruption through specific cleavage of 16S rDNA, and inhibition of
peptidoglycan synthesis. In our knowledge this is the first report of the isolation of bacteriocinogenic
strain of Enterococcus casseliflavus from guava. Enterococcus casseliflavus
ST182Gu, produces a pediocin-like bacteriocin (based on highly homology of
PCR product generated with the primers targeting pediocin PA-1 structural gene) with
activity against several LAB, Listeria spp, and some other human and foodborne
pathogens. No significant differences in growth and production of bacteriocin ST182Gu
were observed when the strain E. casseliflavus ST182Gu was cultured for 24 h in MRS
broth at 20 °C, 30 °C or 37 °C. At this 3 different cultivation temperatures activity was
1.0 x10 9AU/ml against L. ivanovii subsp. ivanovii ATCC19119 and 2.6x10 4AU/ml against
Enterococcus faecium ATCC19443. Low levels of bacteriocin ST182Gu activity (approximately
1.3 x 10 4AU/ml against L. ivanovii subsp. ivanovii ATCC19119 were recorded
after 3h of growth in MRS broth at 37°C and the maximal bacteriocin ST182Gu production
(1.0 x 10 10AU/ml) was recorded in MRS after 27 – 33 h of growth. A decrease in activity
was observed after 33 h of growth and it was reduced to 1.0x10 8AU/ml at 48 h of
incubation. Addition of bacteriocin ST182Gu to exponential or stationary phase cultures
of L. ivanovii subsp. ivanovii ATCC19119 inhibited growth for 24 h. The effect of
bacteriocin ST182Gu on cells of L. ivanovii subsp. ivanovii ATCC19119 was visualised by
AFM and indirectly recorded based on enzyme, protein and nucleotide material leakage.
Bacteriocin ST182Gu is highly active against L. ivanovii subsp. ivanovii ATCC19119
and exhibits a synergetic effect in the inhibition of this surrogate microorganism when
applied together with subletal doses of ciprofloxacin.
* Participation Supported by IUFoST.
Control of Listeria monocytogenes in fresh goat cheese by bacteriocinogenic
strain Lactococcus lactis subsp. lactis DF4Mi or commercial nisin
Nader Furtado, D., Todorov, S.*, Landgraf, M., Destro, M. T. and Franco, B. D. G. M.
University of Sao Paulo , Brazil
Bacteriocins are antimicrobial peptides produced by different groups of bacteria.
Foods can be supplemented with bacteriocins, or by inoculation with the bacteriocinogenic
strains, favouring the in situ production of the bacteriocin. The aim of this
study was to evaluate the capability of the bacteriocinogenic strain Lactococcus lactis
subsp. lactis DF4Mi, isolated from goat milk, to control the growth Listeria monocytogenes
in fresh goat cheese over 10 days of storage under refrigeration, and to
compare the effect that achieved in cheese added of commercial bacteriocin (nisin).
Cheeses were prepared with 10 liters of pasteurized milk acidified with lactic acid
(2.5 % v/v) and added of calcium chloride and commercial rennet, according to the
cheese manufacturer practice. Three sets of cheeses were prepared: one with pasteurized
milk with starter culture strain DF4Mi (10 6 UFC/ml), second with bacteriocin
(-) strain of L. lactis subsp. lactis and other with addition of 12.5 mg/l of pure
nisin (Fluka). L. monocytogenes 701 was added to cheeses during the manufacturing,
in order to obtain 10 3 UFC/g. Appropriative controls such as cheese without any
starter cultures, only L. monocytogenes 701, only strain DF4Mi or only bacteriocin (-
) L. lactis subsp. lactis were prepared. Cheeses were stored at 8 °C and submitted to
counts of lactic acid bacteria and L. monocytogenes every other day for 10 days period.
Results indicate that L. monocytogenes decreased 3-log in the cheeses containing
either bacteriocinogenic L. lactis subsp. lactis DF4Mi or bacteriocin (-) L. lactis
subsp. lactis, showing that inhibition of L. monocytogenes might have occurred due
to another factor than the production of bacteriocin. Analysis of the cheeses containing
nisin demonstrated that incorporation of this bacteriocin at 12.5 mg/l could
inhibit the growth of L. monocytogenes allowing a decrease of 2-log counts during
storage. The present study showed that the use of purified bacteriocin in fresh goat
cheese is more effective in the control of L. monocytogenes than the direct application
of the bacteriocinogenic L. lactis subsp. lactis DF4Mi.
* Participation Supported by IUFoST.

AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
High pressure processing ensures elimination of Listeria monocytogenes
in sliced dry cured ham
Stollewerk, K., Jofré, A., Comaposada, J., Aymerich, T., Ferrini, G., Arnau, J. and Garriga, M.
IRTA, Spain
High pressure processing (HP) is a non-thermal treatment which inhibits or inactivates
microorganisms and it is used to extend the shelf life of food products without
altering their sensorial properties. Listeria monocytogenes can contaminate
different types of meat products, such as dry cured ham, during slicing. The aim of
the present study was to evaluate the behavior of L. monocytogenes in three types
of boned dry cured ham (H1, H2 and H3) produced following different processes.
H1 was tumble salted and smoked; H2 was brine salted and fermented; H3 was
brine salted, fermented and smoked. Afterwards, slices of ham were spiked with a
mixture of 3 strains of L. monocytogenes (ca. 100 CFU/g). Drying was performed by
the Quick Dry Slice process®. Pairs of slices were vacuum packed and half of them
were submitted to a HP treatment of 600 MPa for 5 min. Packages were stored
under refrigeration for 112 days and the evolution of the pathogen was followed by
plating in cromogenic agar and species-specific real time PCR. During storage of
non-pressurized hams the levels of L. monocytogenes progressively decreased in
the three types of ham recording absence in 25 g in H3 after 112 days. Pressurization
produced an immediate reduction of L. monocytogenes to levels below the
plate detection limit (10 CFU/g) in all the hams. In H3 the pathogen was absent
during the whole storage. After 112 days L. monocytogenes was not detected in any
of the samples (absence in 25 g). The composition and processing of dry cured ham
affected the survival of L. monocytogenes. Fermentation and smoking speed up the
decrease of the pathogen, but only the application of a HP treatment enabled to
achieve a product free of L. monocytogenes during the whole storage period.
Inhibition of Listeria monocytogenes by Lactococcus spp. EU2241
in tropical shrimp
Abdoulaye Fall, P.
Ifremer, France
Shrimp is one of the most marketed seafood in the world. Due to its physicochemical
parameters this product is very sensitive to microbial growth such as
pathogens and spoiling microorganisms. Growth of these undesirable flora is often
delayed by the use of classical techniques such as salting, low storage temperature,
modification of atmosphere packaging or use of preservatives. However the
human pathogen Listeria monocytogenes (Lm) that was evidenced in raw and ready
to eat shrimp by some studies can often grow in those conditions and may reach
the tolerated level of 100 Lm g -1. Biopreservation, an alternative technology for
food preservation which consists in inoculating a product with selected bacteria to
eliminate or prevent growth of undesirable microorganisms, may be useful against
Lm. In a previous study, a strain of Lactococcus spp. EU2241 had been isolated
from raw salmon and showed capacities to inhibit in model medium some
pathogens isolated from seafood product. In this study the inhibition capacity of
Lactococcus spp. EU2241 was tested in shrimp artificially contaminated with Lm.
Four batches of cooked shrimp were prepared. 1: sterility control; 2: inoculation
with Lactococcus spp. (10 6 CFU g -1); 3: inoculation with Lm (10 3 CFU g -1); 4: co-inoculation
of Lactococcus and Lm. Shrimp were stored at 8 °C for 31 days under
modified atmosphere (CO 2 /N 2 50-50). When inoculated alone, Lm grew very well
in shrimp reaching its maximum level, 10 9 CFU g -1, in 10 days. An immediate inhibition
of Lm was observed in co-inoculated batch, reaching more than 4 logs at
the end of storage with no sensory changes, confirming the interest of this bacterium
for an application in biopreservation of seafood products against Lm. The
use of Seafood Spoilage and Safety Predictor software to predict the growth of Lm
in shrimp, showed that the lactic acid produced by Lactococcus spp. (2500 ppm)
and pH decrease from 6.60 to 5.91 could not totally explain the inhibition observed
in this study. A chemically defined liquid medium is currently developed to study
the inhibition mechanisms.
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Listeria monocytogenes and Listeria innocua in slaughter line of a swine
meatpacking plant in Rio Grande do Sul State, Brazil
Schittler, L. 1 and Padilha Silva, W. 2
1. UDESC, Brazil
2. UFPeL, Brazil
Listeria monocytogenes is bacterium widely distributed in nature which presents
high capacity to colonize surfaces and form biofilms in food processing plants. It is
the causative agent of listeriosis which may cause a range of diseases from gastroenteritis
to death, being the meat an important vehicle for transmission to humans.
The aim of the present study was to investigate the presence and characterize
L. monocytogenes and Listeria innocua in the slaughter line of a swine
meatpacking plant, located in Missões Region, state of Rio Grande do Sul, Brazil.
For this was evaluated water, cold chambers, carcasses, knives, faeces, hands and
drains and the strains obtained were serotyped and submit to molecular typing
through the analysis by random amplification of polymorphic DNA (RAPD) with
UBC 155 e UBC 127 primers. The occurrence of L. monocytogenes was of 0.95 %
and of L. innocua, 19.4 %, in 105 samples assayed, being the cold chambers the
major point of isolation. Most of the L. innocua strains were not serotypable and,
from those serotypable, the serovars 6a and 6b were prevalent. Only one L. monocytogenes
strain was isolated, from a drain, however it belonged to serovar 4b, an
important serovar in public health, because it is frequently involved in listeriosis in
humans. RAPD of L. innocua strains produced 15 genetic combined profiles, whose
index of discrimination was D=0.94, whereas the serotyping presented low discriminatory
power (D=0.46). There is contamination by L. monocytogenes e L. inoccua
in the slaughter line of the swine meatpacking plant assayed, and the cross
contamination in this plant is considered important, since some L. innocua strains
persist in the local.
Listeria monocytogenes in raw meat products marketed in the city
of Sao Paulo, Brazil: Incidence and counts data for risk assessment
Ristori, C. A. 1, Rowlands, R. E. G. 1, Martins, C. G. 1, Fávero, L. M. 2 and Franco, B. D. G. M. 3
1. Food Microbiology Laboratory, Adolfo Lutz Institute, Sao Paulo, SP, Brazil
2. Food Surveillance, COVISA, Sao Paulo, SP, Brazil
3. Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University
of Sao Paulo, Sao Paulo, SP, Brazil
Listeria monocytogenes is a ubiquitous bacterium frequently found in meat products.
Despite abundance of results on the prevalence of L. monocytogenes in these
foods in different countries, quantitative data on level of contamination required
for appropriate risk assessments are scarce. This study aimed to evaluate the
prevalence and numbers of Listeria monocytogenes in a variety of meat products
available in the local market in the city of Sao Paulo, SP, Brazil. Between May 2008
and July 2009, 552 samples of refrigerated raw meat products (138 beef sausages,
138 pork sausages, 138 ground beef samples and 138 chicken leg samples) were
purchased in 138 local supermarkets, selected after stratification by region and by
random draw. Tests for presence and enumeration of L. monocytogenes were based
on ISO 11290-1:1996/Amd.1:2004 and ISO 11290-2:1998 methods, respectively. L.
monocytogenes was detected in 48.7% of the of meat products samples. The highest
prevalence occurred in ground beef (59.4 %) followed by chicken legs (58 %), beef
sausages (37.7 %), and pork sausages (39.8 %). In most samples (67.4 %), the level of
contamination was lower than 10 2 CFU/g. Counts ranged from < 10 to 5.6x10 2
CFU/g in pork sausages, from < 10 to 1.9x10 2 CFU/g in beef sausages, from < 10 to
8.9x10 2 CFU/g in chicken legs, and from < 10 to 4.5x10 3 CFU/g in ground beef. Despite
the low counts, data on prevalence of L. monocytogenes are relevant for estimating
the risks of listeriosis associated to consumption of meat products in Sao
Paulo, and for establishing science-based intervention strategies aimed at reducing
these risks, specially for pregnant women and immunocompromised individuals.

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Inhibiton of Listeria monocytogenes by Carnobacterium
maltaromaticum in combination with extract of Lippia sidoides Cham.
in cold-smoked surubim fish broth
Barbosa dos Reis, F., de Souza, V. M., Sousa Thomaz, M. R., Pinto Fernandes, L.,
Pereira de Oliveira, W. and Pereira De Martinis, E. C.
Faculdade de Ciências Farmacêuticas de Ribeirão Preto - Universidade de São Paulo, Brazil
Consumption of refrigerated cold-smoked fishes offers risk of listeriosis, the severe
foodborne infection caused by Listeria monocytogenes (Lm). In this work, to
improve the safety ready-to-eat fish, multiple hurdle experiments were carried
out with cold-smoked surubim broth (CSSB) stored at 5 °C for 5 weeks. To inhibit
Lm, it was evaluated the application of a hidroalcoholic extract of the Brazilian
folk plant Lippia sidoides (ELs) (10 µl/ml) in combination with Carnobacterium
maltaromaticum (Cm) C2 and A9b + (bacteriocinogenic strains) and A9b - (nonbacteriocinogenic).
CSSB was inoculated with 10 2 CFU of Lm/ml and/or 10 5 CFU
of Cm/ml, as follow: 1) Lm; 2) Cm C2; 3) Cm A9b -; 4) Cm A9b +; 5) Lm plus ELs; 6)
Cm C2 plus ELs; 7) Cm A9b - plus ELs; 8) Cm A9b + plus ELs; 9) Lm plus Cm C2; 10)
Lm plus Cm A9b -; 11) Lm plus Cm A9b +; 12) Lm plus Cm C2 plus ELs; 13) Lm plus
Cm A9b - plus ELs; 14) Lm plus Cm A9b + plus ELs. Bacterial populations and bacteriocin
titer were determined after 24 h and once a week for up to 35 days. Control
with Lm only, reached ca. 10 8 CFU/ml, but in co-culture with Cm, listerial
population was significantly lower (ca. 10 4 CFU/ml with Cm A9b+ or Cm A9b- and
< 10 2 CFU/ml with Cm C2). Bacteriocin production was observed only for Cm C2
alone and Cm C2 plus Lm, which may explain the stronger inhibitory effect of Cm
C2. ELs only did not inhibit Lm (ca. 10 8 CFU of Lm/ml) and its use in combination
with any Cm strain did not present synergistic effect (ca. 10 4 CFU of Lm/ml). Bacteriocin
production by Cm C2 was not detected in the presence of ELs, suggesting
that ELs can adversely affect the protective culture and decrease the effectiveness
of the biopreservation system studied.
Inhibition of Listeria monocytogenes in cooked ham
by virulent bacteriophages and protective cultures
Holck, A., Schirmer, B. C. and Berg, J.
Nofima Mat AS, Norway
Listeria monocytogenes is found in a number of raw and ready-to-eat (RTE) products,
poultry, seafood and dairy products. RTE products like cooked ham have little
inherent stability against the growth of L. monocytogenes. Several studies show
reduction of L. monocytogenes in food when exposed to phages. Phage particles,
however, become immobilised and are inactivated soon after addition to nonliquid
foods and can thus not prevent later outgrowth of surviving Listeria. Here we have
shown that protective cultures can be used successfully as an additional hurdle
together with phages to reduce growth of L. monocytogenes. Sliced cooked ham
was inoculated with cold adapted L. monocytogenes and exposed to bacteriophages.
The hams were subsequently inoculated with Lactobacillus sakei TH1 protective
culture, vacuum packed and stored at 10 °C. Addition of phages gave an immediate
1-2 log reduction in L. monocytogenes. After 14 – 28 days of storage, an additional 2
log reduction was observed in samples with phages and protective culture compared
to samples with phages alone. The effect of the protective culture was evident
both at 10 and 4 °C. Higher inoculation levels of protective culture gave a
stronger inhibition of L. monocytogenes. The use of phages in combination with
protective cultures is a general method that can potentially be applied to different
products where there is a risk for growth of L. monocytogenes.
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Surface colonization by Listeria monocytogenes: role of the flagella
in the biofilm formation process
Desvaux, M., Briandet, R., Renier, S., Deschamps, J., NCaccia, N., Chafsey, I. and Hébraud, M.
INRA, France
The presence, contribution and/or requirement of exopolysaccharides in the
biofilm formation process in Listeria monocytogenes have not been definitively ascertained.
So far, the flagellum is the only cell surface factor demonstrated as involved
in biofilm formation within this species, even so its exact contribution and
role remain controversial. This prompted us to reinvestigate the role of the flagella
in the course of biofilm formation at growth temperatures relevant to regulation
of genetic expression in L. monocytogenes using both static and dynamic
cultures conditions. Biofilm formation was assayed at early and late stages of the
process using paramagnetic microbeads and crystal violet methods. Cell morphology
and biofilm architecture were determined by confocal scanning laser microscopy
using bacterial cells expressing fluorescent markers. Cells were grown
statically in microtiter plates and in dynamic conditions using flow cells. As revealed
by proteolytic treatments, extracellular/cell-surface proteins rather than
exopolysaccharides are crucial determinants involved in listerial biofilm formation.
While L. monocytogenes was enable to swim at 20 °C, no swarming could be
observed. Kinetics of biofilm formation at 20 and 40 °C revealed that unflagellated
mutant strain formed more biofilm than the wt except at the highest growth
temperature tested. Besides crystal violet method, such results were confirmed
by confocal microscopy in static and dynamic cultures conditions using fluorescently
labelled listerial cells. Biofilm formation was for the first time investigated
over a range of growth temperatures relevant to regulation of genetic expression in
L. monocytogenes species. The use of complementary growth conditions, i.e. static
and dynamic cultures, allowed providing a comprehensive picture of biofilm formation
in this species. In those tested conditions it clearly appeared that flagella
are not essential but rather detrimental to biofilm formation in L. monocytogenes.
The role of sanitizers in controlling Listeria monocytogenes on stainless
steel surfaces: Lessons learned from the 2008 Listeriosis outbreak
Hébert, K., Farber, J. and Pagotto, F.
Health Canada
Proper maintenance and sanitization of food-contact and non-food-contact surfaces
remains a high priority for industry in ensuring a microbiologically safe final
product. Listeria monocytogenes is often present in factory environments and
needs to be controlled to avoid contamination of final product. The national outbreak
of listeriosis caused by contaminated deli-meat raised questions about the
ability of Listeria to survive on stainless steel surfaces. We evaluated the effectiveness
of 11 sanitizers previously and currently used by Canadian industries
against strains of L. monocytogenes. Eleven sanitizers were evaluated using the
American Society for Testing and Materials standardized method (E2197) using
stainless steel disk coupons, according to the recommendations on the labels or
from the manufacturer. A soil load consisting of a microbial suspension, BSA,
mucin and tryptone was used to mimic environmental conditions. Bacteria were
inoculated at 10 7 CFU per coupon, dried and challenged against 50 µl of the suggested
working concentration and exposure time of each sanitizer. The log reduction
was calculated by direct plating. Ten sanitizers met the performance criteria
for L. monocytogenes, achieving a minimal 6-log reduction. There was no significant
difference observed amongst the different active ingredient formulations. A
single, alchohol-based hand sanitizer used by the industry did not satisfy the criteria
when tested on stainless steel coupons. This study demonstrated that sanitizers
are effective in controlling Listeria on stainless steel surfaces when proper
adherences to protocols were maintained. Misuse of sanitizers can allow the organism
to survive and possibly make its way into the final food product.

AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Listeriophage ecology and diversity on dairy farms
Vongkamjan, K.*, Moreno Switt, A., den Bakker, H. C., Fortes, E. D., and Wiedmann, M.
Department of Food Science, Cornell University, Ithaca, NY, USA
Listeria monocytogenes is common in dairy farm environments and contaminated
silage appears to be the major source of L. monocytogenes infections in farm ruminants.
Improved understanding of the ecology and diversity of phages infecting
L. monocytogenes (“listeriophages”) in food associated environments is critical
to evaluate the impact, effects, and potential of phage-based pathogen control
strategies. We collected silage samples over the course of one year on two dairy
farms to perform screening for L. monocytogenes and listeriophages, using standard
methods and representative host strains. L. monocytogenes isolates were characterized
by molecular methods (e.g., sequencing of sigB) and subsequently classified
into lineages I and II. Listeriophages were characterized for host range using
a set of reference strains that represented 13 L. monocytogenes serotypes. Furthermore,
phage genome size was determined by the Pulsed Field Gel Electrophoresis.
Out of 134 silage samples tested (farm 1, n = 81; farm 2, n = 53), six
samples were positive for L. monocytogenes; 43/81 samples from farm 1 and 40/53
samples from farm 2 were positive for listeriophages. Overall 34/134 silage samples
contained >100 PFU/g silage with samples containing up to 2x10 4 PFU/g. For the
majority of phage positive-samples, phages were detected on serotype 4a host
strain. Phage isolates represented considerable host range diversity (12 distinct
lysis groups). Most phages did not lyse lineage I strains, but lysed most lineage II
strains and showed consistent and strong lysis patterns with all lineage III strains.
Phage genome size ranged from 25 to 120 kb. Our data show considerable listeriophage
diversity in dairy farm environments, suggesting that phages play an important
role in the ecology of L. monocytogenes on dairy farms.
* Participation Supported by IUFoST.
Evaluation of curative and preventive decontamination treatments
on Listeria monocytogenes biofilms with a new screening system
Quinon, E. 1, Chamot, S. 1, Groelly, J. 2, Chavant, P. 2, Bernardi, T. 2, Desvaux, M. 1
and Hebraud, M. 1
1. INRA, France
2. BioFilm Control, France
Food safety requires not only interventions on the product but also on all surfaces
of the workshops from the receipt of raw materials until the processing and packaging
of food products. All surfaces can be contaminated by microorganisms able to
adhere, to form biofilms and to disseminate all along the chain. The literature
shows that bacterial cells in biofilm, compared with their planktonic counterpart,
present a greater resistance to hostile environments and particularly to cleaning
disinfection procedures. The efficacy of cleaning disinfection products currently
used for surface decontamination is generally evaluated on planktonic cells, which
does not predict their efficacy on bacteria in biofilm. We have implemented a
screening system, initially developed by the Biofilm Control society to evaluate
bacterial ability to form a biofilm on an abiotic surface (BioFilm Ring Test â), to test
the efficacy of 4 commercial decontamination solutions to detach biofilm from the
surface and to affect the viability of bacterial cells. Three temperatures (10 °C, 20 °C
and 37 °C) and three Listeria monocytogenes strains were used for the tests. The effect
of preventive decontamination treatment of the surface on cell adhesion and
biofilm formation was also assessed. Two out of the 4 products, containing quaternary
ammonium compound (QAC) as biocide, dramatically affected the viability
of sessile cells whatever the temperature while only one out of the two was efficient
to detach adhered cells. The formulation of this last product also contains a
polyenzymatic cocktail. The two other solutions remained inefficient on biofilms.
The QAC + polyenzymatic cocktail was also the only product efficient to prevent or
delay the biofilm formation after a preventive treatment of the surface. The
BioFilm Ring Test â can be used to sreen the efficacy of biocides on bacterial biofilms
in order to improve the microbiological status of the food processing plants.
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AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Pulsed Field Gel Electrophoresis, conventional and molecular
serotyping on Listeria monocytogenes: An European Proficiency
Testing Inter-laboratory Trial
Félix, B., Roussel, S., Tam Dao, T., Asséré, A., Lombard, B. and Brisabois, A.
Agence française de sécurité sanitaire des aliments (Afssa), Laboratoire d’Etudes et de Recherches sur
la Qualité des Aliments et sur les Procédés agro-alimentaires (Lerqap), Maisons-Alfort, France
In Europe, the incidence of listeriosis caused by Listeria monocytogenes is relatively
low, however an increase in cases, which differ depending of the countries,
has been observed since 2000. Different methods are available for first-level characterization
in epidemiological surveillance, in particular, conventional and molecular
serotyping. Regarding fine characterization techniques, macro-restriction
analysis by pulsed-field gel electrophoresis (PFGE) has been described as the most
discriminatory methods for L. monocytogenes subtyping. This method is widely
used by the European National Reference Laboratories (NRLs) according to a
2009 EFSA survey. AFSSA’s Laboratory for Study and Research on Food Quality
and Processing was appointed as Community Reference Laboratory (CRL) for Listeria
monocytogenes in 2006. Recently, the CRL organized a proficiency test in
order to evaluate the ability of the NRLs to perform these three typing methods.
Laboratories were free to choose either one, two or all three methods. The same
panel of six strains was used for all three methods and was sent to the 17 participating
NRLs. This panel included serotypes 1/2a, 4b, 1/2c, 1/2b and 3a strains. Each
laboratory was free to choose the protocols used. All participants followed the
PFGE PulseNet Europe standardized protocol. PFGE migration parameters were
set by the CRL, pattern interpretations were performed following the CRL’s interpretation
criterion. For conventional serotyping and molecular serotyping, 86 %
and 93.5 % of the serotypes obtained were in agreement with the CRL data. Inconsistencies
were mostly explained by differences observed in the use of reagents.
For PFGE, 83 % and 81 % of profiles for AscI-PFGE and for ApaI-PFGE was undistinguishable
of the expected profiles The inconsistencies observed were caused
by slight standardization defaults or in few cases by a fault in the extraction. This
PT trial was a valuable opportunity to improve the typing ability of NRLs and will
allow PFGE pattern exchanges.
Detection of Listeria spp. in raw and pasteurized liquid egg-products
and in the egg-breaking plants environment
Rivoal, K., Fablet, A., Chemaly, M., Salvat, G. and Protais, J.
AFSSA, France
Listeria monocytogenes has been recognized as a human pathogen for decades and
is etablished as an important food-borne pathogen. There is a lack of information
about eggs and egg-products contamination by Listeria spp. The aim of this study
was to detect Listeria spp. in liquid egg-products (whole, yolk and white with or
without salt and/or sugar) collected in 5 egg-breaking plants in north western
France, during one year. To study the seasonal variation, each egg-breaking plant
has been visited during each season. For a raw egg-product sample, a pasteurized
sample has been tested and the egg shells used for theses egg-products have been
swabed to detect Listeria spp. The environment of plants was also sampled with
swabs during the production of the egg-products tested. At the present time, the
project is not achieved. Three seasons have been studied and the last one is now
going on. The environment of the egg-breaking plants seems to be very contaminated
by Listeria spp. when the egg-products and their shells were slightly contaminated
by the bacteria. Moreover, Listeria monocytogenes has been very rarely
isolated in the egg-products as well as in the environment. At the end of the year,
the study will be finished and the results could be analysed.

AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Biofilm formation by Listeria monocytogenes isolates under conditions
that mimic food and digestive tract
Kretli Winkelströter, L. 1, Oliveira, M. A. 2 and De Martinis, E. C. 1
1. Universidade de São Paulo, Brazil
2. Instituto Adolfo Lutz, Brazil
L. monocytogenes is ubiquitous; able to tolerate many adverse conditions found in
foods and can persist in processing plants due to formation of biofilms. L. monocytogenes
can cause severe foodborne infections in immunocompromised people
and in pregnant women. In this work, L. monocytogenes isolates were screened for
ability to form biofilm under diverse conditions. Overnight cultures of 51 isolates
of L. monocytogenes from food, clinical and environmental sources were inoculated
(1:20, v/v) in Brain Heart Infusion Broth (BHI) and under the following conditions:
a) BHI plus 2 % sucrose; b) BHI plus 5 % NaCl; c) BHI plus 0.3 % Oxgall; d)
BHI pH 2 (adjusted with HCl) and e) BHI plus 50 % (v/v) of extract from bacteriocin-producing
cultures of Lactobacillus sakei 1, Leuconostoc mesenteroides 11A or
Enterococcus faecium 130. Aliquots of inoculated broths (200µl) were transferred
to wells of a polystyrene 96-wells microtiter plate and incubated at 37 °C/24h.
Wells were washed 3 times with Phosphate Buffered Saline (PBS) and each well
was stained with 200µL of 1 % (v/v) crystal violet aqueous solution for 15 min.
Plates were washed 3 times with PBS and wells were destained with 200µL of 95 %
ethanol. Concentration of crystal violet present in the destaining solution was
measured at 595 nm (CV-OD 595 ) and directly correlated with biofilm formation. L.
monocytogenes ATCC 19115 was used as parameter of comparison for biofilm formation.
L. monocytogenes ATCC 19115 presented CV-OD 595 in the interval of 0.69-
0.87 under all conditions tested. CV-OD 595 >1.1 was observed for approximately
9.8 %, 1.9 %, 7.8 % and 19.6 % for L. monocytogenes isolates incubated respectively in
BHI only and under presence of sucrose, NaCl and Oxgall, indicating a higher ability
to form biofilm. In BHI pH 2 and BHI plus 50 % (v/v) of extract from bacteriocin-producing
cultures, L. monocytogenes isolates did not show higher differences
in biofilm formation in comparison with L. monocytogenes ATCC 19115 (0.70-0.81
CV-OD 595 ). L. monocytogenes isolates showed different abilities to form biofilm
under different conditions probably due to modulation of gene expression, which
will be further studied.
Biofilm formation of Listeria monocytogenes EGDe depends
on temperature and nutrient availability
Auchter, M.*, Endres, J., Waidmann, M. S. and Riedel, C. U.
Institute of Microbiology and Biotechnology, University of Ulm, Ulm, Germany
Bioluminescent in vivo imaging revealed that in mice, Listeria monocytogenes colonizes
the gall bladder prior to systemic spread. Moreover, bacterial cells of L.
monocytogenes isolated from the gall bladder display chaining morphology, a phenotype
associated with biofilm grown cells. Thus, biofilm formation in the gall
bladder could be a strategy of this important pathogen to protect, establish and
multiply itself at a site inaccessible to the components of the immune system prior
to causing systemic infections. To investigate the mechanisms of biofilm formation,
we tested L. monocytogenes EGDe and an isogenic agrD deletion mutant for
their ability to form biofilms under different conditions. Biofilm formation was
tested in a standard microtiter plate assay at different time points during biofilm
growth of EGDe, varying temperatures (20, 30 and 37 °C) and in full-strength BHI
(rich broth) as well as in 10-fold diluted BHI medium (nutrient limited). Biofilm
formation at 37 °C was poor in rich broth and could be significant enhanced when
cells were grown under nutrient limitation. Deletion of the agrD gene resulted in
defective biofilms in 10-fold diluted, as well as in full-strength BHI. The biofilm
formation of the ∆agrD mutant could also be restored to wild type levels if reconstituted
cell-free supernatant (BHI added to spent EGDe supernatant, pH adjusted
and filter sterilized) of EGDe wild type cells grown in full-strength BHI was added
to the ∆agrD strain. Additionally, DDAO staining and DNAse treatments revealed
a potential role for extracellular DNA in the initial stages of biofilm formation.
* Participation Supported by IUFoST.
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AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Antimicrobial activity of lactococcal and enterococcal strains
isolated from artisanal products from North West of Italy toward
Listeria monocytogenes
Dal Bello, B., Rantsiou, K., Ambrosoli, R., Zeppa, G. and Cocolin, L.
Department of Exploitation and Protection of the Agricultural and Forestry Resources, University of
Turin, Italy
Listeria monocytogenes is a foodborne pathogenic Gram-positive bacterium that is
widely distributed in soil, sewage, fresh water sediments and effluents, and is frequently
carried in the intestinal tract of animals and humans. Biocontrol of L.
monocytogenes by bacteriocin-producing lactic acid bacteria or by bacteriocin extracts
has attracted great attention in recent years and new preservation strategies
to control growth of L. monocytogenes have been developed, including the application
of the bacteriocin nisin (Food and Drug Administration, 1988). In this work,
we have investigated the potential role as bioprotection agents of autochthonous
lactococcal and enterococcal strains isolated from fresh and fermented artisanal
products (cheese and meat) of Piedmont region (North West of Italy) determining
their antimicrobial spectrum of activity towards different foodborne spoilage and
pathogenic microorganisms. Bacteriocin-producing strains were identified by molecular
methods and genetic determinants encoding the antimicrobial proteins
were targeted by PCR. Thirty-nine strains of Lactococcus lactis exhibited inhibition
towards L. monocytogenes NCTC 10527 and most of them (26 strains) showed
the presence of the genes responsible for the nisins A and Z production. Regarding
Enterococcus spp., 31 strains showed inhibition activity towards L. monocytogenes
NCTC 10527 and the presence of the genes responsible for the enterocins A and P
production was determined. It is interesting to underline that for some strains it
was not possible to identify any known bacteriocins. In this study a high incidence
of bacteriocin producing strains was observed. In the future, the possible use of
these active strains could be a new way to ensure safety of foods.
Plant-based strategies for Listeria monocytogenes control in foods
Paparella, A., Serio, A., Chaves-Lopez, C. and Di Pasquale, F.
University of Teramo, Italy
Essential oils (EOs) and plant extracts are considered a natural and effective alternative
to chemical preservatives. Considering the complex chemical composition
of EOs, their antimicrobial action is not likely attributable to one single mechanism,
although bacterial cytoplasmic membrane seems to be a specific target.
The authors evaluated the antimicrobial activity of thyme, oregano and cinnamon
EOs against Listeria monocytogenes strains, isolated from the smoked salmon industry
and from meat products; these strains showed variable levels of antimicrobial
resistance, belonged to different serotypes and had heterogeneous molecular
profiles. Thyme and oregano EOs were particularly effective, with Minimal Inhibitory
Concentrations (MICs) correlated with strains biodiversity. Automatic
turbidometry was applied to evaluate the effects of EOs on cell physiology; the results
documented a lag phase extension and a decrease of maximum growth rate
and maximum growth value. The antibacterial effect was evident even after a short
contact time with the cells. Using flow cytometry combined with fluorescent techniques,
the authors demonstrated that membrane disruption is the primary inactivation
mechanism of thyme and oregano oils. A different mechanism might be involved
in cinnamon EO, acting on cells enzymatic activity. Further studies, carried
out by means of EPR (Electronic Paramagnetic Resonance), highlighted the effect
of oregano EO on membrane fluidity and order, variable with increasing EO concentrations.
These findings prove that food biopreservation with EOs involves various
mechanisms of action and may be considered a safe and effective measure to
control Listeria monocytogenes growth, although with different effects depending
on strains biodiversity.

AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Scientific studies for survival of Listeria monocytogenes
in dairy products and its application in practice
Cabanova, L., Škuntova, O. and Kantikova, M.
State Veterinary and Food Institute, Dolny Kubin, Slovakia
European reference laboratory for Listeria monocytogenes in Paris has prepared
together with some National reference laboratories from the European Member
States the scientific study for Listeria to prove the producer that his products are
safe till the end of the shelf- life. In our study we have developed studies for the
producer of two different dairy products after several positive findings of Listeria
in the final products. As a first product the raw sheep cheese has been selected
and as a second one the steamed cheese products have been analyzed. In the first
stage the physico-chemical characteristics has been determined (a w , pH) and than
the subsamples has been prepared and artificially contaminated with a mixture
of two reference Listeria strains and third isolated strain from the previous positive
sample. After inoculation treated samples has been stored at 5.8–6.2 °C incubator
till the end of the shelf-life and during this period 2-3 times has been analyzed
(a w , pH, Listeria detection and enumeration has been determined). At the
end of the shelf – life the growth potential has been calculated and the Listeria
numbers at the beginning and at the end of the shelf- life has been given.
The effect of chilling temperatures on the virulence
of Listeria monocytogenes isolates with different origins
Neves, E. M. 1,2, Silva, A. C. 1*, Louro, P. 3,4, Ferreira-Dias, S. 4 and Brito, L. 1
1. CBAA/ Departamento de Botânica e Engenharia Biológica, Instituto Superior de Agronomia,
Technical University of Lisbon, Portugal
2. Instituto Superior de Estudos Interculturais e Transdisciplinares (ISEIT), Campus Universitário
de Almada, Instituto Piaget, Portugal
3. Instituto Nacional dos Recursos Biológicos, IP, Portugal
4. CEER/ Departamento de Agro-Indústrias e Agronomia Tropical, Instituto Superior de Agronomia,
Technical University of Lisbon, Portugal
Although the chilling and freezing of food products are increasingly used by the
food industry, there are few studies on the effect of these temperatures on the virulence
of Listeria monocytogenes. The influence of 1, 7 and 30 days of storage at
chilling (7 °C) and freezing temperatures (-20 °C and -76 °C) on the virulence of
19 L. monocytogenes isolates was evaluated in this study. The isolates selected according
to their genetic diversity and different initial levels of virulence were from
frozen (n = 5), refrigerated (n = 3), refrigerated ready-to-eat (n = 5) and ready-toeat
foods (n = 1), from humans (n = 2), from dairy environment (n = 1) and reference
strains (n = 2). In the case of the chilling assays, the cells were kept in physiological
buffer and culture medium was added just before infection. After the
respective storage, the isolates were used to infect HT-29 cell monolayers in a
plaque forming assay (pfa), immediately or after thawing the bacterial suspensions
at room temperature. The pathogenic potential of the strains was expressed
as the mean log of the number of plaques formed (log pfa), and one-way or multifactorial
ANOVA of the counting data was carried out. The results showed that the
refrigeration temperature (7 °C) acted more significantly (P < 0.05) on the decreasing
of the virulence potential of all isolates, although time didn´t show a significant
effect on this loss of virulence. In relation to freezing temperatures (-
20 °C and -76 °C), the decrease in virulence was not as significant, although the
temperature of -76 °C tended to be more conservative of the initial virulence. Better
elucidation of which refrigerated food matrices allow growth of the pathogen
may help to evaluate the real risk of the presence of Listeria in food.
* Participation Supported by IUFoST.
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AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
How to improve a sampling plan in order to better assess
L. monocytogenes contamination on diced bacon at the plant
Bergis, H. 1, Commeau, N. 1, Zuliani, V. 2, Cornu, M. 1, Beaufort, A. 1 and Garry, P. 2
1. AFSSA, France
2. IFIP, Institut de la filière porcine, France
Food Business Operators (FBO’s) are legally responsible for the safety of their food
products (Regulation CE 2073/2005). The regulation does not specify the frequency
of sampling/ testing and it is up to the FBO’s to decide the appropriate
level of sampling/testing to help validate and verify their food safety management
plans. In this scope, sampling plans are useful tools to control and to assess the
contamination of a given foodborne pathogen at the plant and also to help the FBO
to take decisions. In the present study, a production of diced bacon in a French
plant was monitored to assess the contamination of this product by Listeria monocytogenes.
The steps of the process are: tumbling, steaming, dicing, and packaging.The
aim of this study was to try, through the elaborated sampling plan, (i) to establish
a link between the prevalence of L. monocytogenes in the pork breasts and
in diced bacon; (ii) to determine whether the levels of contamination of the pork
breasts and in the diced bacon are related; (iii) to estimate the incidence of the
tumbling step and of the dicing step on the final product contamination.The food
process was studied from the raw material (pork breasts) to the final product
(diced bacon) and the steps possibly involved in the contamination identified. An
experimental stratified random sampling plan was constructed in two steps of the
process: after tumbling (a important step), and after packaging. 106 sampled tumbling
breast and 86 packages of diced bacon were analysed. Detection and enumeration
of L. monocytogenes were performed on 100cm2 of the pork breast and on
100g of diced bacon. The lactic acid bacteria (LAB) were also enumerated. From
the obtained data on contamination (prevalence and level of contamination of L.
monocytogenes and the level of LAB), it appears that the tumbling step has an homogenisation
effect on the contamination and sampling after this step could be a
good indicator of the presence of L. monocytogenes in diced bacon. It has been
shown that over a certain level of contamination of the breast, the diced bacon
from the same batch are also contaminated with L. monocytogenes.
Contamination of Listeria monocytogenes in a cold-smoked
pork processing plant using brining injections
Berzins, A. 1, Silins, I. 1 and Korkeala, H. 2
1. Faculty of Veterinary Medicine, Latvia University of Agriculture; University of Helsinki, Latvia
2. University of Helsinki, Finland
Contamination of L. monocytogenes was studied in a cold-smoked pork processing
plant using brining injections. Overall prevalence of L. monocytogenes in coldsmoked
pork products during a 5-year period was 26 %. Environmental sampling
combined with PFGE subtyping and serotyping was applied to investigate the genetic
diversity of L. monocytogenes in the brining facilities and alongside premises.
A total 183 samples were collected for contamination analyses, including samples
of the product at different stages during manufacture (n = 136) and
environmental samples (n = 47) covering most of the manufacturing surfaces during
a 3-month period. Overall, the prevalence of L. monocytogenes in raw pork before
processing was 18 %. The prevalence of L. monocytogenes in pork increased
to 60 % after being in brining area and brining machine. Two different L. monocytogenes
PFGE types belonging to serotypes 1/2a and 1/2c were recovered from the
different sites of the brining machine (feeding teeth and smooth surfaces), thus
causing persistent contamination of RTE cold-smoked pork products over period
of 5-years. In addition, brining machine harboured two PFGE types belonging to
serotypes 1/2a and 4b, which were found on different contamination sites: feeding
teeth, smooth surfaces and spaces of the machine. Brining injections increased L.
monocytogenes contamination in finished RTE cold-smoked pork products and
processing environment. Brining area, and specifically, brining machine should
be subjected for disassembling and extensive cleaning and disinfection to eliminate
any persistent contamination with L. monocytogenes.

AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Effects of GRAS products on growth of Listeria monocytogenes during
cold storage of salmon fillets
McCarthy, S. and Johnson, D.
Food and Drug Administration, USA
Microbial growth can affect the quality and safety of prepared, processed, and
ready-to-eat (RTE) meat, poultry, and seafood. Pathogenic bacteria, including Listeria
monocytogenes (Lm), can be resistant to preservatives, sanitizers, and antibiotics.
The presence of Lm on RTE products results from cross-contamination
from contact surfaces in food processing plants. This study examined the ability of
three GRAS products to inhibit the growth of Lm on salmon fillets during cold
storage. Twenty-five-g portions of raw salmon fillets were inoculated externally
with six log 10 CFU Lm 1a1/g. Inoculated fillets were incubated at 25 °C for 20 minutes
and at 4 °C for two hours to allow attachment. Filets were then treated with
sterile deionized water (SDW; control) and 3 GRAS products (IONAL TM LC, MOstatin
TM VS, levulinic acid; 1:5 w/v) at 25 °C for 5 min. Control and treated filets
were analyzed immediately after exposure (T0) and after storage at 4 °C for up to
12 weeks. Fillets were shaken in wash buffer and cell counts in washates were determined
by spread plating on chromogenic agar. Numbers of Lm increased by
one log 10 during 9 weeks of cold storage of fillets treated with SDW. In contrast,
MOstatin TMVS reduced the numbers of Lm by two logs 10 during storage for 9
weeks. IONAL TMLC and levulinic acid prevented the growth of Lm for up to four
weeks. Each of the GRAS products reduced densities of salmon bacterial flora by
two to four logs 10 at two weeks compared to SDW; however, no difference was observed
at 12 weeks. The use of GRAS products could prevent an increase in numbers
of Lm that are associated with cross-contamination in processing plants.
Heavy-metal and detergent resistance of Listeria species isolates from
milk processing environments
Doijad, S.*, Garg, S. and Barbuddhe, S. B.
ICAR research complex for Goa, India
Listeria monocytogens is an important food-borne pathogen responsible for varied
clinical forms in animal and humans. The resistance of Listeria strains to cadmium,
arsenic and quaternary ammonium compounds was studied and it was correlated
with resistance to quaternary ammonium compounds used as disinfectants in the
food-processing industry. Limited information is available on the prevalence of resistance
among isolates from the environment of food-processing plants. In this
study, a total of 27 Listeria species isolates from the milk processing plants were
included. Out of 27, 14 (51 %) found to be Listeria monocytogenes, 12 (44 %) were L.
innocua and 1 (3 %) isolate was of L. ivanovii. All the isolates were subjected for determination
of the resistance to cadmium, arsenic and quaternary ammonium disinfectant
(benzalkonium chloride (BC)). Isolates were not inhibited at 40µg/ml of
cadmium chloride, while 12 (44 %) of them grew upto 200µg/ml. Isolates grew well
in 40µg/ml of sodium arsenite while 3 of them could tolerate 200µg/ml. The isolates
could grew at 1µg/ml of BC while only 7 could tolerate 5µg/ml. The amount of
BC tolerated by these isolates are well above the amount used in milk processing
environment. There was no co-relation found between resistance of isolates to cadmium,
arsenate and BC, all isolates were independently resistance to metal and detergent.
It was interesting to note that none of the isolates contained plasmid. This
suggests that resistance against metals and detergent in Listeria spp. is not mediated
by the plasmid and need further investigations. Our findings suggest that the
milk processing environment constitute a reservoir for L. monocytogenes and other
Listeria species. Resistance to heavy metals and quaternary ammonium disinfectant
is of significance to food processing industry.
* Participation Supported by IUFoST.
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AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Detection of Listeria monocytogenes in lettuce sold at markets
and supermarkets in Porto, Portugal
Noronha, L., Magalhães, R., Silva, J. and Teixeira, P.
CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal
Listeriosis is a severe infection caused by Listeria monocytogenes particularly
among the elderly, very young and immunocompromized individuals and has also
been associated with late-term miscarriages in pregnant women. The high incidence
of L. monocytogenes in foods and the high fatality rate associated with listeriosis,
has contributed to L. monocytogenes being considered a public health hazard
and a continuing source of loss to food processors due to the large number of voluntary
and obligatory recalls. Listeriosis outbreaks have been linked to the consumption
of raw vegetables including lettuce. According to the European Food
Standards Agency, lettuce supports L. monocytogenes growth and can be related
with the transmission of listeriosis. Twenty different lettuce samples collected in
markets or supermarkets located in the North of Portugal, were analyzed for the
presence of L. monocytogenes by the VIDAS method. Of the tested lettuce samples,
30 % were positive for the presence of L. monocytogenes. Three isolates were collected
from each of the positive samples. They were serotyped by Multiplex PCR
and it was possible to distinguish two serogroups: 33 % belonged to serogroup 4b –
4d – 4e and 67 % to 1/2c – 3c. The present results reinforce the importance of
washing raw vegetables thoroughly before eating and preparing green salads and
vegetable dishes shortly before eating in order to reduce the risk of listeriosis.
Preliminary analysis of structure and chemical composition of extracellular
polymeric substance produced by Listeria monocytogenes
Nwaiwu, O.*, Lad, M., Davis, A., Foster, T. and Rees, C.
Univeristy of Nottingham, UK
It is generally believed that, while Listeria monocytogenes is capable of forming
biofilms, it does not produced extensive amounts of extracellular polymer substances
(EPS) that contribute to adhesion and persistence in the environment. We
present here evidence that under specific growth conditions L. monocytogenes is
capable EPS synthesis and this does promote surface adhesion. After EPS extraction,
analysis by NMR, ATR-FTIR and SEM indicated that the material is high molecular
weight and the structure of Listeria EPS is different from both polysaccharide
and poly-g-glutamic acid (an EPS produced by a range of Gram-positive
bacteria). Further amino acid analysis also ruled out this material being a polymer
of other amino acids, while elemental energy dispersion x-ray analysis showed
very low nitrogen content with carbon and oxygen the predominant elements. The
as yet unidentified polymer has unusual physical properties, and scanning electron
microscopy (SEM) showed rapid absorption of moisture by the EPS as relative
humidity increased. Hence it is likely that this polymer will also contribute to desiccation
tolerance of the bacterium.
* Participation Supported by IUFoST.

AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Ecology and persistence of Listeria monocytogenes strains in fermented
meat sausage processors from the Northern region of Portugal
Ferreira, V. 1, Barbosa, J. 1, Vongkamjan, K. 2, Moreno Switt, A. 2, Hogg, T. 1, Gibbs, P. 1,
Wiedmann, M. 2 and Teixeira, P. 1
1. CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal
2. Cornell University, USA
In this study, 202 Listeria monocytogenes isolates were recovered from product
samples representing 7 processors of Alheira, a traditional fermented meat sausage
from the Northern region of Portugal. Samples were collected from each processor
in separate dates, either at retail establishments or at processing plants. Isolates
were characterized by molecular serotyping and DNA macrorestriction analysis
by pulsed field gel electrophoresis (PFGE). Characterization by PFGE suggested
persistence of particular strains over time in the 7 different processors, the apparent
time of persistence ranged from 14 to 32 months. While a number of studies
have evaluated associations of different strain characteristics, including biofilm
formation etc., with strain persistence in processing plants, no convincing and
consistent evidence for specific strain characteristics that facilitate establishment
of persistence have been found so far. We thus investigated whether phage susceptibility
and presence of lysogenic prophages in L. monocytogenes may be associated
with strain persistence in the environment. A subset of 41 L. monocytogenes
isolates representing sporadic and persistent PFGE patterns was screened for
lysogeny. Twenty six prophages where induced and their lytic spectrum against
the 41 strains of L. monocytogenes was investigated. Lysogens included both sporadic
and persistent PFGE types. While molecular serogroup D (4b, 4d, and 4e)
isolates were more susceptible to phages as compared to serogroup A (1/2a and
3a) or B (1/2b, 3b, and 7) isolates, there was no evidence for differences in phage
susceptibility between persistent and sporadic strains. While our findings support
that L. monocytogenes serotypes differ in phage resistance, strain persistence does
not seem to be associated with enhanced phage resistance.
Biofilm formation and survival of L. monocytogenes and slaughter house
bacteria on surfaces at relevant environmental conditions
Langsrud, S., Møretrø, T. and Heir, E.
Norwegian Institute of Food, Fisheries and Aquaculture Research, Norway
Adhesion, biofilm formation and survival of microorganisms on food contact surfaces
are of concern in the food processing industry. Surface associated bacteria
can lead to cross contamination of food and have serious consequences for human
health and food quality. Persistence of Listeria monocytogenes in the food processing
environment is a well known food safety concern. Also, other environmental
bacteria may cause problems when persisting in the food industry. A better
knowledge on the ability of dominant bacteria to produce biofilm and survive on
food contact surfaces under relevant conditions is therefore important. We have
studied biofilm formation and survival of L. monocytogenes and environmental
bacteria isolated from surfaces in a meat slaughter house. Bacteria were isolated
after cleaning and disinfection and prior to slaughter on a regular working day
and identified by 16S rDNA sequencing. Selected isolates from dominating genera
and L. monocytogenes were tested for their ability to form biofilms under various
temperature conditions in a microtiter plate assay. We also studied survival on
stainless steel under controlled temperature and humidity conditions. The susceptibility
of surface adhered bacterial cells to four commercial disinfectants commonly
used in the meat industry was investigated. Predominant bacteria in the
slaughtering line belonged to the genera Pseudomonas, Serratia, Acinetobacter,
Citrobacter, Aerococcus, Kocuria and Staphylcococcus. The data indicated low
biofilm forming abilities of L. moncytogenes at relevant food industry temperatures,
while other environmental bacteria showed variable biofilm formation at
12 and 20 °C. Gram-positive bacteria showed excellent survival on stainless steel
during incubation at 12 °C, 70 % relative humidity. The bactericidal effects of disinfectants
on bacteria adhered to surfaces were low (< 3 log kill) for all tested disinfectants.
In conclusion, survival of bacteria after cleaning and disinfection in
the meat industry can be explained by resistance to disinfectants when dried on
surfaces and either ability to survive at dry conditions or biofilm formation in the
presence of water.
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AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Control of L. monocytogenes by lysozyme combined with olive leaf
extract in edible pullulan film coated on chicken breast fillets
Handan Baysal, A.*
Izmir Institute of Technology, Department of Food Engineering, Urla, Izmir, Turkey
In recent years, naturally occurring antimicrobial and antioxidant compounds
have been preferably employed in meats because of their potential health benefits
and safety compared with synthetic preservatives. The objective of this study was
to evaluate the antimicrobial effect of olive leaf extract (OLE) and lysozyme added
to pullulan film coatings against Listeria monocytogenes in raw chicken breast meat
stored aerobically under refrigeration (4 °C). The effectiveness of these compounds
in a meat model system was evaluated by surface inoculation (approximately
10 6 CFU/g) of L. monocytogenes onto chicken breast fillets. Fresh chicken
breast fillets used in this research was purchased from a local supermarket. The inoculated
raw chicken breast fillets were treated before storage by dipping into 5 %
(w/v) pullulan film-forming solutions with and without the addition of antimicrobial
agents: (a) deionized water, (b) 5 % (w/v) pullulan + 20 % (w/v) water extract
of OLE, (c) 5 % (w/v) pullulan + lysozyme (d) 5 % (w/v) pullulan + 20 % (w/v)
water extract of OLE + lysozyme) for 10 min at 20 °C. After removal of excess moisture,
the samples were stored at 4 °C. The inhibitory effects of OLE and lysozyme
containing edible coatings were evaluated for 14 d. In the meat system, the L.
monocytogenes population was decreased effectively after 14 d at 4 °C. This research
has demonstrated that the use of an edible film coating containing natural
extracts or the application of a lysozyme solution is a promising means of controlling
the growth and recontamination of L. monocytogenes on raw chicken
breast meat stored under refrigeration.
* Participation Supported by IUFoST.
Evaluation of antilisterial activity by lactic acid bacteria
Borges, S., Barbosa, J., Albano, H., Silva, J. and Teixeira, P.
CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal
Listeriosis is an infection with a high morbidity and mortality, caused by Listeria
monocytogenes. It occurs primarily in elderly patients, immunocompromised individuals,
pregnant women and their neonates. Some lactic acid bacteria (LAB)
are able to produce antimicrobial compounds with activity against several
pathogens, such as organic acids (lactic and acetic acids), hydrogen peroxide, antimicrobial
enzymes and bacteriocins. The objective of this work was to evaluate
the antimicrobial activity of selected LAB against L. monocytogenes. Thirty-five
isolates of LAB, available at the culture collection of Escola Superior de Biotecnologia,
demonstrated inhibitory activity against 29 clinical isolates of L. monocytogenes.
To characterize this antilisterial activity the antagonistic spectrum of each
LAB culture, neutralized cell-free supernant and neutralized cell-free supernant
treated with catalase or trypsin were investigated. All isolates of LAB showed antibacterial
effect probably due to the production of bacteriocins. The bacteriocin
activity (AU/mL) was determinated for three serotypes of L. monocytogenes (1/2a,
1/2b, 4b) and the activity varied between 800 and 6400 AU/mL. According to this
study, the use of bacteriogenic LAB can be important to control L. monocytogenes
of clinical origin.

AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Molecular methods to assess Listeria monocytogenes route
of contamination in a dairy processing plant
Cocolin, L., Alessandria, V., Dolci, P. and Rantsiou, K.
Univeristy of Turin, Italy
In this study we investigated the occurrence of Listeria monocytogenes in a dairy
processing plant during two sampling campaigns in 2007 and 2008. Samples represented
by semi-finished and finished cheeses, swabs from the equipment and
brines from the salting step, were subjected to analysis by using traditional and
molecular methods, represented mainly by quantitative PCR. Comparing the results
obtained by the application of the two approaches used, it became evident
how traditional microbiological analysis underestimated the presence of L. monocytogenes
in the dairy plant. Especially samples of the brines and the equipment
swabs were positive only with qPCR. For some equipment swabs it was possible to
detect a load of 10 4-10 5 cfu/cm 2, while the ISO method employed gave negative
results both before and after the enrichment step. The evidences collected during
the first sampling year, highlighting a heavy contamination of the brines and of
the equipment, lead to the implementation of specific actions that decreased the
contamination in these samples during the 2008 campaign. However, no reduction
in the number of L. monocytogenes positive final products was observed, suggesting
that a more strict control is necessary to avoid the presence of the
pathogen. All the isolates of L. monocytogenes were able to form biofilm, and, interestingly,
considering the results obtained from their molecular characterization
it became evident how strains present in the brines, were genetically connected
with isolates from the equipment and from the final product, suggesting a
clear route of contamination of the pathogen in the dairy plant. This study underlines
the necessity to use appropriate analytical tools, such as molecular methods,
to fully understand the spread and persistence of L. monocytogenes in food producing
companies.
Persistence of L. monocytogenes in artisanal cheese producing plants
Almeida, G., Santos, I., Magalhães, R., Barbosa, J., Hogg, T. and Teixeira, P.
CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal
The consumption of raw milk or raw milk products has caused several listeriosis
outbreaks resulting in several hundred cases. This highlights the risk of these products
and led public health officials to recommend that raw milk and dairy products
prepared from raw milk should not be consumed by susceptible populations, particularly
pregnant women. The presence of L. monocytogenes in cheeses has been
widely reported in the literature over the years. The prevention of Listeria contamination
is difficult to achieve in raw milk cheeses. Also, contaminated raw milk
can contaminate the plant environment and some strains can colonize and persist
for long periods. The present work aimed to characterize L. monocytogenes isolates
recovered between 2004 and 2007 from an artisanal ewe’s raw milk cheese producing
plant. PFGE using restriction enzymes AscI and ApaI was performed in
forty isolates: 18 from cheese, one from raw milk, 20 from environmental sites, and
one from whey. Six combined pulsotypes were obtained, one of them aggregating 20
isolates. This is an evidence of the presence of a resident clone with a persistence
time estimated in at least, 14 months. The profile obtained from cheese isolates
was also encountered in isolates from ewe’s raw milk and from raw milk reception’s
floor suggesting that raw milk could be the source of contamination. Control L.
monocytogenes in the environment plays an important role in reducing its presence
in foods, which is necessary to reduce the incidence of listeriosis.
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AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Occurrence of Listeria monocytogenes in food products collected in
Portugal from retail establishments and food plants
Mena, C., Carneiro, L., Santos, I., Magalhães, R., Almeida, G. and Teixeira, P.
CBQF / Universidade Católica Portuguesa – Escola Superior de Biotecnologia, Porto, Portugal
Listeriosis is an infection caused by the bacterium Listeria monocytogenes. The
ingestion of contaminated food with this microorganism may cause serious health
problems for consumers. During two years (2007 and 2008) the presence of L
monocytogenes was evaluated in a total of 1476 food samples, collected from retail
and food plants. The detection of the microorganism was performed using the automated
VIDAS system and the positive results were confirmed following the ISO
11290 standard. L. monocytogenes was detected in 134 (9.1 %) of the analyzed samples.
Most of positive samples were from foods normally submitted to heat treatment
before consumption such as pré-cooked foods (30.0 %, 12/40; pizza, pasta,
rissois, etc) and fermented meat products (20.6 %, 33/160; farinheira, alheira,
morcela, bacon), from raw products (16.0 %, 58/362; raw meat, vegetables and fish),
and from “ready-to-eat” foods: fermented meat products (8.8 %, 10/114), vegetables
salads (1.1 %, 2/174), ready cooked meals (4.5 %, 6/134), cheeses (4.6 %, 12/262)
and fresh cheese (1.3 %, 1/80). The occurrence of the microorganism in ready-toeat
foods is of more concern. L. monocytogenes grows at refrigeration temperatures
and could achieve levels of contamination that can cause disease. Also, foods
that will have a heat treatment at consumer’s home could represent a hazard if
cross contamination of food items that will be consumed without any further step
of destruction occurs.
Listeria monocytogenes biofilms grown at 12 °C showed reduced
susceptibility to sanitizers
Lourenço, A., Machado, H.* and Brito, L.
CBAA – Departamento de Botânica e Engenharia Biológica, Instituto Superior de Agronomia, Technical
University of Lisbon, Portugal
Listeria monocytogenes may form biofilms on food contact surfaces of difficult sanitization
which may lead to recurrent contamination of food products. The eradication
of biofilms can only be achieved by using adequate hygienization routines
which will ultimately ensure food safety. The biofilm forming ability of four L.
monocytogenes strains from different origins, cheese, dairy environment and from
human cases of listeriosis was evaluated, either in pure culture or in co-culture
with Pseudomonas aeruginosa, at 37 °C and 12 °C using the Calgary Biofilm Device®
(CBD). The minimum biofilm eradication concentration (MBEC) was determined
for four commercial dairy sanitizers (one alkyl amine acetate based, T99;
two chlorine based, T66 and DD and one phosphoric acid based, BP). Co-culture
biofilms had an average total population of 7 to 8 Log 10 CFU/peg. P.aeruginosa
was the dominant species, either at 37 °C or at 12 °C, representing 99 % of the total
CFU/peg. L. monocytogenes biofilms grown, either at 37 °C or 12 °C, although with
different incubation times (24 hours and 7 days, respectively) reached a similar
cell density (6 Log 10 CFU/peg). Nevertheless, the biofilms produced at 12 °C were
generally less susceptible to the sanitizers than when produced at 37 °C. One of
the strains (3880) retrieved MBEC values of 30720 µg/ml and 16000 µg/ml for
T99 and BP, respectively. These values were above the maximum in-use recommended
concentrations (T99 – 29700 µg/ml and BP – 11500 µg/ml) for these
agents. The growth in co-culture also proved to be relevant regarding disinfectant
susceptibility, as the co-cultures were generally less susceptible than L. monocytogenes
pure cultures. The MBEC values obtained for the chlorine based agents
were never over recommended in-use concentrations which may indicate a more
efficient ability to eradicate biofilms, if in-use conditions are met.
* Participation Supported by IUFoST.

AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Modelling growth of Listeria monocytogenes in cheese as function
of environmental variables
Sand Rosshaug, P. and Hallberg Larsen, M.
Faculty of Life Science, Department of Veterinary Pathobiology, Denmark
This project is part of a larger project with the purpose to develop a predictive
model of growth of the pathogen Listeria monocytogenes in a dairy chain producing
blue/white mould cheese. This project focuses on the growth kinetics of L. monocytogenes
as function of environmental variables. Temperature, water activity, pH,
lactic acid, NaCl, and O 2 are environmental factors that impact the growth kinetics
of L. monocytogenes. The growth kinetics was investigated for 3 strains of L.
monocytogenes that has been found in the dairy industry: ATCC 19111, ATCC 19113,
ATCC 19115. Different growth media were tested: broth, milk, and blue/white
mould cheese. The growth kinetics of L. monocytogenes as function of these variables
was formulated as an ordinary differential equation using Cardinal Parameters
to describe the influence of the environmental factors. The growth kinetics
was applied in a predictive mathematical, deterministic model of L. monocytogenes.
Finally the growth kinetics of L. monocytogenes was investigated using a
stochastic predictive model taking into account the stochastic nature of some of
the inputs including the lag.
Characterization of anti-Listerial bacteriocin produced by Lactobacillus
plantarum ST8SH, a strain isolated from Bulgarian salami
Todorov, S. D. 1* and Lemos Vaz-Velho, M. 2
1. Universidade de São Paulo, Faculdade de Ciências Farmacêuticas, Brazil
2. Escola Superior de Tecnologia e Gestão, Instituto Politécnico de Viana do Castelo, Portugal
Strain ST8SH, isolated from Bulgarian salami, was identified as Lactobacillus plantarum
based on biochemical tests, sugar fermentation reactions (API50CHL), PCR
with species-specific primers and 16S rDNA sequencing. Strain ST8SH produces a
3.0kDa class IIa bacteriocin, active against Listeria monocytogenes, Listeria innocua,
Streptococcus caprinus, Streptococcus spp., Lactobacillus casei, Lactobacillus
curvatus, Lactobacillus salivarius, Lactobacillus pentosus, Enterococcus mundtii,
Enterococcus faecalis and Lactococcus lactis subsp. lactis. No change in activity
was recorded after 2 h at pH values between 2.0 and 12.0, and after treatment at
100 °C for 120 min or 121 °C for 20 min. The mode of activity against L. innocua, L.
monocytogenes is bactericidal, resulting in cell lyses and enzyme- and DNA-leakage
and was visualised by atomic force microscopy. The highest level of activity (25600
AU/ml) was recorded when cells were grown at 37 °C or 30 °C in MRS broth (pH
6.5). Peptide ST8SH adsorbs at low levels (400 AU/ml) to producer cells. High cell
numbers of L. plantarum ST8SH and L. innocua LMG13568 were recorded at beginning
when co-cultured. However, the cell numbers of L. innocua LMG13568 decreased
from 1.6x10 4 CFU/ml to 2.5x10 2 CFU/ml in 12 h and to undetectable levels
after 24 h. Plantaricin ST8SH production was stimulated by presence of L. innocua
LMG13568 (102 400 AU/ml). Similar results were obtained with addition of 10 %
autoclaved overnight culture of L. innocua LMG13568 to MRS growth media on
production of plantaricin ST8SH. Based on the genetic approach strain ST8SH
harbours associated genetic determinants for production of a variation of the well
known plantaricin 423. Future purification of the produced bacteriocin need to
be performed to determine if Lactobacillus plantarum ST8SH produces this bacteriocin
(plantaricin 423 – like) or harbour more then one bacteriocin operons.
* Participation Supported by IUFoST.
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AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
EFSA’s proposal for an EU-wide retail survey on Listeria monocytogenes
in selected categories of ready-to-eat food products
Boelaert, F., Felício, T. and Makela, P.
EFSA, Italy
The European Food Safety Authority and its Task Force on Zoonoses Data Collection
were requested by the European Commission to provide a proposal for technical
specifications on an EU-wide retail survey on Listeria monocytogenes in selected
categories of ready-to-eat (RTE) food products that should take place during
the whole year of 2010. The proposed technical specifications focus on sampling
those categories of RTE food in which the highest L. monocytogenes contamination
have been observed in the European Union (EU): soft and semi-soft cheeses,
smoked and gravad fish, and heat-treated meat products that are handled after
heat treatment. Sampling of these RTE food categories would be targeted at retail
outlets serving the final consumer, with catering and wholesale establishments
excluded. Food products are suggested to be tested at the end of the shelf-life and
additionally in the case of smoked and gravad fish, immediately after sampling. At
the Community-level a total of about 3,000 samples should be taken for each RTE
food category, per analyses stage. A specific number of samples is proposed to be
proportionally allocated to the Member States according to the size of their human
populations. Standardised analytical L. monocytogenes detection and enumeration
methods are proposed to be employed in the analyses of samples. In addition,
water activity and pH values are to be measured in the smoked and gravad fish.
This proposal for a Community-specific survey would only allow estimation of the
L. monocytogenes prevalence at the Community level. A modelling and simulation
approach should be applied in the analyses of the results so that the effectiveness
of the implementation of Community L. monocytogenes criteria may be assessed. A
similar model-based approach will also be used to estimate the growth potential of
L. monocytogenes in smoked and gravad fish.
Ripening conditions: an asset to control L. monocytognes in cheeses
Callon, C., Picque, D., Corrieu, G. and Montel, M.-C.
INRA, France
The EC regulations for dairy products require the absence of L. monocytogenes
output production with a possible derogation at < 100cfu/g if it is demonstrated
that there is no evolution during storage until consumption. To identify the microbial
populations of cheeses and environmental factors that may be barrier to L.
monocytogenes in the European project Truefood an ecological approach was preferred
over a process of screening of strains. This approach based on the following
steps: 1) selection of milk with anti-Listeria properties, 2) identification of microbial
communities and simplifications of their composition, 3) cheese-making experiments
in different conditions of ripening. This strategy was successfully applied
to the inhibition of L. monocytogenes in cheese core. Indeed, a microbial
community of raw milk, composed of 7 strains of lactic acid bacteria (Lactobacillus
and Leuconostoc), in synergy with bacteria called ripening Gram positive (Staphylococcus,
Arthrobacter, Brachybacterium, Microbacterium, Corynebacterium, Brevibacterium)
inhibited L. monocytogenes at the same extent than the complex community.
The decrease of relative humidity from 98 to 93 % can also act as a barrier
against L. monocytogenes, especially at the surface of cheeses with or without microbial
consortium. It led also to an increase of dry matter at the surface of cheeses
and a decrease of pH which may contribute to the inhibition of L. monocytogenes.
Interestingly, at the beginning of ripening (8 days), a temperature of 13 °C, by
favouring the growth of Lactobacillus and Leuconostoc composing the microbial
consortium, was more effective than 9 °C for inhibiting Listeria. This can be explained
by the highest galactose metabolism and acid production. On the contrary
in the control with only S. thermophilus, the ripening temperature of 13 °C
favoured the growth of L. monocytogenes. The ripening conditions reasoned according
to the composition of microbial communities could be a promising strategy
in the control of L. monocytogenes.

AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 126–177, 187–198
Deterministic and stochastic behavior of Listeria monocytogenes
suspended cells or detached from stainless steel surfaces during
cheese manufacturing
Belessi, C.-E. A. 1, Gounadaki, A. S. 1, Arapakh, S. 1, Schvartzman, S. 2, Jordan, K. 2
and Skandamis, P. N. 1
1. Agricultural University of Athens, Greece
2. Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
Growth probability and kinetic models for Listeria monocytogenes in response to
multiple hurdles occurring during cheese manufacturing are mainly focused on
suspended L. monocytogenes cells. This study aimed to compared: (i) the
growth/no growth interface of L. monocytogenes cells attached on stainless steel
(SS) surfaces, or in suspension, within adjusted media and (ii) the behavior of
planktonic and detached Listeria cells during manufacturing and ripening of two
popular Greek cheeses: Feta and Graviera. A multi-strains composite of L. monocytogenes
isolates from cheese, factory and farm in Greece and Ireland, were grown
in TSBYE, MRD, Milk, Feta and Graviera cheese in the presence of SS coupons
(2x5cm 2) for 3d at 20 °C, to obtain the following inocula: planktonic cells (P), and
cells detached from the SS coupons (D). Detachment took place by the bead vortexing
method. For growth/no growth evaluation P and D cells were inoculated in
TSBYE, adjusted to 5 pH (6.8-4.8) by lactic acid and at 4 a w (0.945-0.995) by NaCl.
For evaluation of L. monocytogenes kinetics in cheese, P and D cells were inoculated
at three simulated stages of Feta and Graviera manufacture: in pasteurized
milk, after cutting the curd and after the first ripening. The growth of D cells
slightly delayed compared to P cells while it was more affected by a w than pH. On
cheese, L. monocytogenes survived throughout the ripening at low levels. The differences
in probability of growth of single cells for both inocula (P and D) were
assessed by stochastic approaches. Furthermore, PFGE analysis resulted that 91 %
of the cells of any tested condition belonged to the cheese factory isolate. The results
may address safety implications relevant to the potential of attached cells to
proliferate, whereas data may contribute to filling data gaps on risk assessment
of L. monocytogenes isolates from the dairy industry.
Prevalence of Listeria monocytogenes in game meat
Atanassova, V.
Institute of Food Quality and Food Safety, Germany
Listeria spp. are widespread in the environment. Foods of animal origin like raw
meat, milk, soft cheese and smoked fish are often contaminated by Listeria. During
slaughtering and food processing, these bacteria can distribute and establish as
specific contaminants in difficult to clean areas of the plant and the processing
environment. Freshly shot game meat can be readily contaminated during the cutting
and trimming steps. Listeria endures most non heat processing steps and will
be present in packaged retail meats. Chill storage for long periods of time can result
in a considerable increase in numbers. In this study 797 (roe deer, red deer
and wild boar) samples of wild game from freshly shot whole carcasses and 481
samples from packaged game meat stored under chill conditions (+ 4°C) were analyzed
for the presence of Listeria spp. and Listeria monocytogenes. Standard methods
according to ISO 11290 were applied. Enrichment of meat samples was performed
first in half Fraser, followed by full Fraser broth. Enrichment cultures were
streaked to OCLA plates. In total 7.3 % (n=58) of the meat from whole carcasses
was positive for Listeria spp., with a total of 39 (4.9 %) samples being contaminated
by Listeria monocytogenes. There was no difference in the prevalence in
meat from different game species. Chill stored packaged game meat showed higher
prevalence rates of 20.6 % (n=99) Listeria monocytogenes positive samples. The
results show that game meat can be contaminated by Listeria monocytogenes.
Prevalence was lower at the beginning of the meat processing in carcasses still unskinned,
while during elongated storage the rate can be higher in chill stored meat
cuts. To reduce the risk of infection for the consumer, proper cooking is advised
during meal preparation.
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Relationship between pathogenic profile and in vitro biofilm formation
capacity of Listeria monocytogenes strains isolated from meat, fish and
processing plants
Meloni, D., Mazza, R., Marceddu, M., Piras, F., Mureddu, A. and Mazzette, R.
Department of Animal Biology, Faculty of Veterinary Medicine, University of Sassari
In the present survey, the relationships between pathogenic profile and in vitro
biofilm formation of 106 Listeria monocytogenes strains having no epidemiological
correlation and isolated from different environmental and food sources, were analyzed.
The isolates were grouped into different categories based on the source of
isolation: swine and poultry carcasses (14 and 13 % respectively), ground meat
(7 %), fermented sausages (10 %), raw and smoked salmons (9 and 4 % respectively),
swine slaughterhouse environments (3%), fermented sausage and smoked salmon
processing plants environments (24 and 16 % respectively). The quantitative assessment
of the in vitro biofilm formation was carried out by using a microtiter
plate assay with spectrophotometric reading (OD 620 ). The cut-off value (ODc) was
equal to three time the standard deviation of the negative controls plus the average
OD reading for the same controls. The strains were divided up into four categories,
based on their ability to form biofilms: no biofilm producers (OD≤ODc), weak producers
(ODc

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Minimum Biofilm Eradication Concentration (MBEC)
of different antimicrobials on Listeria monocytogenes
and Salmonella enterica biofilms
Rodrigues, D. 1, Teixeira, P. 1, Oliveira, R.1, Ceri, H. 2 and Azeredo, J. 1
1. Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do
Minho, Campus de Gualtar, Braga, Portugal
2. Biofilm Research Group, Department of Biological Sciences, University of Calgary, Canada
Inadequate disinfection of food processing environments contributes to foodborne
disease outbreaks, particularly those concerning L. monocytogenes and Salmonella
enterica. The ability of these bacteria to adhere and form biofilms on several surfaces
makes sanitation more difficult and challenging, reason why susceptibility
tests must cover not only planktonic cells but also adhered cells and biofilms.
Through MBEC assessment using Calgary Biofilm Device (CBD), this work aimed
at comparing the performance of four antimicrobials on L. monocytogenes and Salmonella
enterica biofilms and the disinfection efficiency between strains and
species. Three L. monocytogenes strains (994, 1562 and CECT 4031 T) and five S.
enterica strains (355, CC, NCTC 13349, LT2 and ATCC 140285) were used. Biofilms
were grown on CBD for 24 hours, in Mueller-Hinton Broth, at 37 °C with shaking at
125 rpm. Disinfection was performed with sodium hypochlorite, benzalkonium
chloride, hydrogen peroxide and triclosan, while bacterial death was assessed by
standard plate method on Trypticase Soy Agar after sonication. Results showed
that sodium hypochlorite had the lowest MBEC values while triclosan had the
worst performance, since no S. enterica biofilm eradication was achieved even at
the maximum concentration used. It was also found that, except for hydrogen peroxide,
MBEC values for L. monocytogenes biofilms were similar or inferior to those
found for S. enterica. Significant differences on minimum survival biomass results
were also observed between strains of the same species. Summarizing, this work
has pointed out chlorine agents as the most effective on disinfecting biofilms of
both species used and revealed a higher susceptibility of L. monocytogenes biofilms
to disinfection in general when compared with S. enterica biofilms. Moreover, not
only interspecies but also intraspecies variability were found to influence disinfection
efficacy.
Examination of the ability of adherence, biofilm formation and sensitivity
to some disinfectants of different Listeria monocytogenes strains
Milanov, D. 1, Vidić, B. 1, Petrović, J. 1, Bugarski, D. 1 and Ašanin, R. 2
1. Scientific Veterinary Institute “Novi Sad”, Novi Sad, Republic of Serbia
2. Faculty of Veterinary Medicine, Republic of Serbia
The objective of this work was to examine the capabilities of 14 Listeria monocytogenes
strains to attach on glass and to form biofilm on stainless steel surfaces.
The strains originated from animals (8 strains), food (4 strains) and feed (1 strain).
The referent strain was a human isolate (ATCC 19115). The number of L. monocytogenes
was determined after 3-hours of attachment and after 48-hour incubation
in tryptone soy broth at 25 °C and 37 °C by the use of standard count technique
on blood agar from tenfold dilution. Three days old biofilm formed on glass surfaces
of the selected L. monocytogenes strains was treated with paracetic acid and
phenol disinfectants for 5 and 10 minutes. On the stainless steel surface the
biofilms were formed during 7 days of incubation in a tryptone soy broth supplemented
with 0.6 % yeast extract (TSB-YE) at the temperature of 25 °C. The developed
structures were examined using scanning electron microscopy. For all the
examined L. monocytogenes strains the number of attached bacteria to glass slides
for 3 hours of incubation in static conditions at 25 °C and 37 °C ranged from 10 2-
10 4 cfu/cm 2. After 48 h of incubation the number of cells that grow on glass slides
did not depend on initial attachment and for all the strains it was 10 5-10 7 cfu/cm 2.
Among tested Listeria monocytogenes strains, significant differences in terms of
their ability to form biofilm were found. Seven of 14 investigated strains of Listeria
monocytogenes did not form biofilm, and only individual bacterial cells were distributed
over the stainless steel surface. The strains classified as biofilm producers
formed structures of different appearances, from a uniform, confluent monolayer
of bacterial cells to individual large, three-dimensional cell aggregates. L. monocytogenes
cells attached to glass surface expressed higher resistance to disinfectants
than the cells in suspension.
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Evolution of Listeria monocytogenes contamination
in poultry production: from the farms to the processing levels
Mansour Chemaly, M., Toquin, M.-T., Courtillon, C., Le Nôtre, Y., Rivoal, K. and Fravalo, P.
AFSSA, France
This investigation is a part of a large french program aiming to assess and update
data regarding L. monocytogenes in poultry flocks (laying hens, broilers, turkeys)
and the impact of their introduction in the slaughtering process. Samples were
collected from the flocks (5 bootswabs) which were followed up to the slaughterhouses
where sampling consisted of swabs from the environment (before and after
cleaning and desinfecting procedures), caeca and products (neck skins and fillets).
The isolation of L. monocytogenes was performed according to the NF EN
ISO11290-1 method and the identification included serotyping. RFLP-PFGE was
carried out in order to establish the clonal relationships and to trace the contamination
between the farms and the slaughterhouses. 32 % of the flocks were tested
positive for Listeria monocytogenes. The serotyping of L. monocytogenes strains
showed that the majority belonged to the type 1/2a which presented high genetic
diversity (Simpson Index: 0.95). At the slaughterhouses, the contamination varied
between 4 and 26 % of the total sampling. Positive samples were found in the environment
after cleaning and desinfection and the same isolates were found in the
environment between the slaughtering of other batches and on the products.
Strains found at the farm level were clearly different from those found at the
slaughterhouses. Our work highlighted the spreading of L. monocytogenes in poultry
flocks, residual and cross contamination at the slaughterhouses. Despite a high
level of faecal contamination at the farm level, no relation could be established
with the contamination of slaughterhouses. This suggests that the primary production
is not the main source of contamination of poultry products. Efforts
should be focused on the slaughterhouses where inefficient C&D procedures and
cross contamination were the main identified sources of contamination.
A regular survey of Listeria in ready-to-eat foods (2004 – 2009)
Furtado, R., Loreto Campos, M., Correia, C., Ferreira, I., Maia, C., Rosa, N., Santos, S.,
Santos, M. I. and Saraiva, M.
Departamento de Alimentação e Nutrição, Instituto Nacional de Saúde Doutor Ricardo Jorge, I.P.,
Lisboa, Portugal
Listeria monocytogenes is one of the most important pathogens found in food. The
consumption of food products contaminated with this bacterium can cause listeriosis,
a disease with a high mortality rate that can affect especially vulnerable
groups. The ubiquitous nature of Listeria, its resistance to several environmental
conditions and the ability to grow at refrigeration temperatures, promote the occurrence
of contamination in any stage of the food chain. Taking simple precautions
like thoroughly cooking foods, respecting the chill chain, proper washing
fruit and vegetables eaten raw and washing hands repeatedly, reduce contamination
chances. The main activity of our Laboratory, it is the control of the microbiological
quality of ready-to-eat foods served in canteens. This regular survey includes
the evaluation of the lay out of the physical facilities based on Reg. (EU)
n. º 852/2004 and Codex Alimentarius, and the collection of food samples and
swabs in surfaces and utensils. This work evaluate the presence of Listeria monocytogenes
(VIDAS LMO2 Bio 12/11-03/04) and quantify the level of Listeria spp.
(ISO 11290-2:1998/Amd:2004), in food samples collected in the establishments
surveyed by INSA Lisboa, between 2004 and 2009. In a total of 5450 ready-to-eat
foods analysed, Listeria monoytogenes was present in 73 samples, in which 8.2 %
exceeded the 100 cfu/g limit. Considering that daily thousands of meals are consumed
by risk populations, such as those of kindergarten, schools, hospitals and
homes for the elderly, and that our positive results correspond to 58.9 % of samples
collected in these type of establishments, they could stand as a useful tool, demonstrating
that if additional precautions are not implemented, Listeria monocytogenes
can be a potential risk for these particularly susceptible groups, even at concentrations
less than 100 cfu/g.

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Incidence of Listeria monocytogenes in Queijo Fresco
Rosa, N., Campos, L., Correia, C., Ferreira, I., Furtado, R., Maia, C., Santos, S.,
Cunha, C. I. and Santos, M. I.
Departamento de Alimentação e Nutrição, Instituto Nacional de Saúde Doutor Ricardo Jorge, I.P.,
Lisboa, Portugal
Listeria monocytogenes is a ubiquitous bacterium responsible for inumerous cases
and outbreaks of listeriosis in humans, usually transmitted by the consumption
of contaminated foods, mainly those “ready-to-eat”. Being “Queijo Fresco” a readyto-eat
product, it’s one of those which have driven Listeria monocytogenes to be a
major concern to Public Health. It was the purpose of this study to evaluate the
presence of Listeria monocytogenes in “Queijo Fresco” from a number of brands
with the most commercial significance in the Lisbon area. The “Type of Commercial
Presentation” was also studied for its influence in the occurrence of the microrganism
in the above mentioned food products. A total of 125 samples were examined
for the presence of Listeria monocytogenes. Secondary enrichments, in
Fraser broth, were analysed by the mini-VIDAS LMO® (bioMérieux, Durham,
France), enzyme-linked fluorescent immunoassay method for Listeria monocytogenes
detection. Positive samples were confirmed by isolation on ALOA® (AES
CHEMUNEX) selective agar followed by biochemical characterization with API
LISTERIA® (bioMérieux, Durham, France). Statistical analysis was performed
using the Statistical Package for the Social Sciences (SPSS v13.0) software. Of 125
samples, 13 (10.4 %) were positive for Listeria monocytogenes. It was statistically
proved that the Commercial Presentation influences the presence of Listeria
monocytogenes, being the cheeses sold packed less contaminated than those sold
unpacked (p = ,049). This study demonstrates that Listeria monocytogenes is present
in “Queijo Fresco” from a range of brands commercialized in Lisbon. The contamination
observed, represents a potential risk for the Portuguese consumer due
to his natural taste for this product.
Risk factors for Listeria monocytogenes contamination
in French broiler flocks
Aury, K., Le Bouquin, S., Toquin, M.-T., Petetin, I., Le Nôtre, Y., Allain, V.,
Fravalo, P. and Mansour Chemaly, M.
AFSSA, France
Despite a decreasing incidence of Listeriosis in France since 1999, Listeria monocytogenes
continue to be a public health concern. In order to update data relating
to L. monocytogenes in France in poultry production, an epidemiological study was
conducted in broiler chicken flocks between October 2005 and September 2006
aiming to identify the potential risk factors associated to the presence of L. monocytogenes.
142 broiler chicken flocks were included in this study. The L. monocytogenes
status of these flocks was based on 5 samples of bootswabs per farm. The
flocks were considered positive if at least one sample was tested positive for L.
monocytogenes. Information on potential risk factors was collected by questionnaire
at the same time as sample collection. The association between characteristic
management practices and L. monocytogenes status was assessed by logistic regression.
The prevalence of L. monocytogenes contamination in these flocks was
31.7 %. The risk of L. monocytogenes contamination was increased when farmer
did not respect the principle of the two areas (clean and dirty) at the poultry house
entrance. The absence of spraying at the first disinfection and/or pest control of
the poultry house before the arrival of the next flock was found to increase the
risk of being infected. When litter storage was not protected and when farm staff
take care of the other broiler chicken houses of the holding, the risk of L. monocytogenes
contamination increased significantly. For the watering system, pipettes
without recuperator were found to be associated with a higher risk of contamination
than pipettes with recuperator or drinkers. This study brings new insights
for the risk management of L. monocytogenes infection in broiler flocks.
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Portuguese sushi: is it contaminated with L. monocytogenes?
Mendes, D., Furtado, R., Maia, C., Correia, C., Campos Cunha, I., Pedroso, L. and Santos, M. I.
Instituto Nacional de Saúde Doutor Ricardo Jorge
Healthier nutritional lifestyles and cultural globalization have popularized the
consumption of ready-to-use products of seafood, like sushi, that were previously
restricted to oriental countries. Sushi is a traditional Japanese food, mostly composed
of rice and raw fish. The sushi trend involves a higher consume of raw fish,
particularly among young, urban people including pregnant women. Although fish
is considered a healthy food, as with other animal products, consumption of raw
muscle incurs potential health risks such as ingestion of pathogenic bacteria. Additionally,
the preparation practices involved in the production of sushi has the
potential to allow contamination, namely with Listeria monocytogenes, the agent
of listeriosis, an infection that targets mainly pregnant women (and their fetuses),
children, the elderly and immunocompromised individuals. In spite of the number
of cases per annum is relatively low, these infections can be acute, with mortality
up to 30 %. Due to the seriousness of clinical manifestations and high rates of mortality
in populations at risk, control and prevention of this disease, represents an
important challenge to sanitation authorities and deserves the attention of food
microbiologists and health professionals. In this study, 90 samples of sushi collected
from different establishments in Lisbon, Portugal, were analyzed for their
microbiological status and the prevalence of pathogenic bacteria. For L. monocytogenes
detection the ISO 11290-2:1998/Amd 1:2004 method was performed. The
results obtained showed that all samples were negative for L. monocytogenes and
just one of them revealed the presence of Listeria seeligeri. Although the number of
samples studied was small, we believe this study provides important information
concerning the incidence of L. monocytogenes in Portuguese sushi and also evidences
that the consumption of this type of food probably is not a major problem
in relation with this pathogen.
Prevalence of Listeria monocytogenes throughout the production
process of Parma ham: tracing contaminations from slaughterhouses
to the final product
Prencipe, V. A. 1, Rizzi, V. 1, Iannetti, L. 1, Serraino, A. 2, Calderone, D. 3, Rossi, A. 4, Morelli, D. 1,
Marino, L. 1 and Migliorati, G. 1
1. Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy
2. University of Bologna - Veterinary Medicine, Italy
3. Consorzio del Prosciutto di Parma, Italy
4. Centro Ricerche Produzioni Animali S.p.A., Italy
In order to evaluate Listeria monocytogenes prevalence and contamination levels
throughout the Parma Ham production chain, 774 swine carcasses were traced
along the whole process until the final product. Analyses were carried out on samples
originated from the same carcass but taken at three different points of the
process (as carcass, fresh ham and dry-cured ham). Fecal samples were also taken
from 498 carcasses (about 2/3 of the whole sample). The prevalence of Listeria
monocytogenes, calculated at 2.9 % in the carcasses swabbed in slaughterhouses,
increased up to 12.5 % in fresh hams sampled when entering the manufacturing
plants and eventually fell to 2.0 % at the end of the production chain (de-boned
and packaged dry-cured hams). Listeria monocytogenes was isolated from only one
fecal sample (prevalence 0.2 %), corroborating the evidence of the low importance
of primary production as a source of contamination from this bacteria. Contamination
levels ranged from 0.10 to 0.33 MPN/cm 2 in carcasses, 0.04 to 2400 UFC/g
in fresh hams, 0.04 to 100 UFC/g in dry-cured hams sampled at the end of the production
chain. Only the 3.2 % (n = 3) of contaminated fresh hams came from contaminated
carcasses, no one among the 14 contaminated dry-cured hams came
from contaminated fresh hams. Certainly, cutting was the stage with the highest
contamination risk throughout the Parma Ham production process. However, beside
the sharp reduction of contaminations subsequent to dry-curing, seasoning
environment should be addressed as the critical step of the whole process for the
contaminations found in the final product. The significant differences between
plants corroborates the importance of the processing environment as source of
contamination from Listeria monocytogenes. Further biomolecular analyses are
needed in order to confirm the marginal role of transferring contaminations between
different stages of the production chain and to assess the presence of persistent
strains inside the processing plants.

AREA // D // Strategies for prevention and control of Listeria monocytogenes
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Effect of the inoculum size on growth of L. monocytogenes
in dices of poultry breast
Lardeux, A.-L., Gnanou-Besse, N., Doux, C. and de Courseulles, E.
AFSSA, France
Traditionally the microbiological safety of foods has been established via challenge
tests. Since the contamination level attained by Listeria monocytogenes may depend
on the initial bacterial concentration, it is important to take into account this effect
when performing challenge tests. To date, there are, to our knowledge, few studies
which have examined its impact in food. The effect of the inoculum size on growth
of L. monocytogenes in a matrix of dices of poultry breast has been studied. This matrix
is interesting because it is located near the interface growth/no growth of L.
monocytogenes, it has a natural microflora and contains lactic acid. Challenge tests
have been carried out for three batches, according to technical guidance document
of Community Reference Laboratory for Listeria monocytogenes. Thus, the fastest
strain of L. monocytogenes has been selected by a study of strains growth with
potassium lactate at different concentrations. Then, L. monocytogenes growth studies
were carried out at 8 °C, using two contamination levels (1 cfu/g and 100 cfu/g),
in vacuum-sealed bags or under modified atmosphere (50 % nitrogen – 50 % carbon
dioxide). Follow-ups of natural microflora (mesophilic and lactic) have been
performed, as well as the measurement of physical-chemical parameters (pH, a w
and lactic acid). L. monocytogenes was able to grow in all conditions. However,
growth was higher under vacuum packaging conditions, and with a high inoculation
level. Moreover, important differences were observed between batches. In parallel,
an important growth of background microflora was observed, which probably had a
strong impact on L. monocytogenes growth.
Investigation into the mechanisms of detergent induced changes
in disinfectant susceptibility of attached Listeria monocytogenes
Walton, J., Hayes, R., Protheroe, R., Hill, D. and Gibson, H.
University of Wolverhampton, UK
The control of Listeria through effective cleaning and disinfectant strategies is
crucial to maintaining the safety of food. The aim of this work is to investigate the
effect of detergent treatments on the susceptibility of attached Listeria monocytogenes
to subsequent disinfectant treatments. While the detergents were prepared
to working concentrations, the disinfectants were lower than the recommended in
use concentration to allow quantification of changes in susceptibility. Results so
far have shown that L. monocytogenes, attached to stainless steel surfaces, became
significantly less susceptible (up to 3.5 log10 difference) to benzalkonium chloride
(BAC, 0.005 % v/v) following treatment with the anionic detergents sodium
alkyl sulphate (SAS, 0.2 % v/v), sodium dodecyl sulphate (SDS, 0.2 % w/v) and
sodium lauryl ether sulphate (SLES 0.2 % v/v). L. monocytogenes also became significantly
less susceptible (0.4 log10 difference) to sodium dichloroisocyanurate
(NaDCC, 0.0008 % w/v) following treatment with SAS but significantly more susceptible
(1 log10 difference) following treatment with SLES. The non-ionic detergent,
fatty alcohol ethoxylate (FAE, 0.1 % v/v), had no effect on susceptibility to
either BAC or NaDCC. The changes in susceptibility may be due to effects on cell
membrane permeability, cell surface hydrophobicity and/or reduced uptake due to
efflux. Flow cytometry using the fluoresceine propidium iodide revealed significant
increases in cell membrane permeability by SAS and FAE while no change
was observed with SDS. Hydrophobic interaction chromatography showed that L.
monocytogenes became less hydrophobic following treatment with SAS and SDS
but FAE had no effect. Current work using ethidium bromide has shown a reduction
in fluorescence following treatment with SAS and SDS suggesting that the detergents
may trigger an efflux mechanism resulting in reduced uptake of disinfectant.
To conclude, detergents can influence the susceptibility of L. monocytogenes
to BAC and NaDCC which does not appear to be related to changes in cell membrane
permeability. However, susceptibility does appear to correlate with changes
in cell surface hydrophobicity and enhanced efflux.
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Prevalence of Listeria monocytogenes in raw milk sold
at vending machines in Abruzzo region
Prencipe, V. A., Scattolini, S., Sperandii, A. F. and Migliorati, G.
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy
A survey was carried out in Abruzzo region for assessing the microbial quality of
raw milk sold at self-service automatic vending machines. From January to October
2008, 299 raw milk samples, collected at 20 distributors, were tested to detect
Listeria monocytogenes. Moreover, the presence of the compulsory information
for consumer and the observance of the proper storage temperature were
verified. 31 samples (10.5 %) positive for Listeria monocytogenes were found, taken
from 6 distributors supplied by 3 farms. 29 over the 31 positive samples were collected
at 4 vending machines supplied by only one farm. All the Listeria monocytogenes
strains were identified as serotype 4 b. The resistance to two antimicrobials
was detected in 26.6 % of the strains tested showing two different patterns
(OXCC, OXL), in 74.2 % to three antimicrobials with only one profile (OXCCL).
PFGE analysis identified a single pulsotype specific for each farm, whose samples
had been found contaminated by Listeria monocytogenes. The combination of the
resistance patterns and restriction profile of the 29 samples isolated from A farm,
identified three different subtypes. The persistence of Listeria monocytogenes in
raw milk samples, supplied by A farm, confirmed that an inadequate hygiene management
exposes the consumer to a real risk of infection by Listeria monocytogenes.
The absence of information for the consumer and the lack of cold chain
maintenance during the distribution step could increase this risk. Therefore, it is
necessary that farmers and distributors’ managers apply continuously the own
check and its efficacy should be verified by the Competent Authority implementing
specific surveillance plans. However it is fundamental to inform the consumer
correctly and on continuous basis about the potential risks associated to food
products and on the right handling procedures during transport, storage and treatment
of food products.
Characterization of Listeria monocytogenes strains isolated from soft
and semi soft cheeses sampled at retail level
Acciari, V., Torresi, M., Migliorati, G., Di Giannatale, E., Semprini, P. and Prencipe, V.
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy
The aim of this study was to characterize the 47 Listeria monocytogenes strains
isolated during a survey carried out on cheeses sampled at retail level. Five semisoft
and soft cheeses (gorgonzola, taleggio, asiago, crescenza and brie) were selected
among the higher consumption dairy products in Italy and the most frequently
contaminated by Listeria monocytogenes. Each strain was serotyped, tested
for susceptibility to antimicrobials and pulsotyped using AscI and ApaI Pulsed
Field Gel Electrophoresis (PFGE). The main serotypes were 1/2a (76.6 %) and 1/2c
(21.3 %), the 1/2b was isolated in only one sample. The antimicrobial resistance
patterns showed that most Listeria monocytogenes strains were resistant to
oxacillin (97.6 %), lincomicin (80.9 %) and clindamycin (78.7 %). The resistance to
two antimicrobials with two different resistance patterns (OXCC, OXL) was detected
in 17 % of the strains tested, to three antimicrobials with one resistance
profile (OXCCL) in 70.2 %. No strains were sensitive to all antimicrobials tested.
Combination of AscI and ApaI macrorestriction patterns yielded 11 different pulsotypes
clustering in three groups. Two main pulsotypes were found by grouping
21.3 % and 57.4 % of the strains isolated. Evaluation of PFGE profiles showed no relationship
between pulsotypes and cheese type, manufacturer or retail outlet.
Temporal distribution of the prevailing pulsotypes showed a persistent profile
throughout most of the study period, except from August to September, when a
different pulsotype was found. Therefore the temporal variation in the prevalence
of specific strains, as reported in this survey, could be the consequence of factors
able to influence the product contamination pattern. Large scale studies will contribute
to assess the dynamics of Listeria monocytogenes contamination.

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Preliminary report on the organisation of a food microbiology
proficiency testing program as a tool to guarantee the equivalence
of the US and IT official control systems
Di Giannatale, E., Marfoglia, C., Prencipe, V., Salini, R., Migliorati, G. and Ricci, L.
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Italy
After Listeria monocytogenes was isolated in products imported from Italy, in 2002
the Italian Ministry of Health developed an integrated approach to assure the
equivalence between the Italian and the US official control systems. A key part of
the ongoing program has been carried out thought an ad hoc laboratory proficiency
testing with the aim of monitoring the technical skills of the official laboratories in
charge of testing Listeria monocytogenes on meat products to be exported in the
USA. This study describes the organization of a proficiency testing and the protocol
experimented for preparation and verification of samples consisting in food matrix
with a basic microflora contaminated with Listeria monocytogenes at two different
level of contamination. The proficiency testing is an indispensable tool to assess
the performance level of a testing laboratory and to demonstrate its reliability at
national and international framework. In our specific context, the proficiency program
has also concurred as one of the effective measures to guarantee the equivalence
within the certifications issued by the Italian and US official laboratories.
High nisin susceptibility of Listeria spp. wild-type strains isolated
from dairies with traditional cheese preservation in Portugal
Pintado, C. M. B. S1,2 and Ferreira M. A. S. S. 2
1. Escola Superior Agrária, Instituto Politécnico de Castelo Branco, Castelo Branco, Portugal
2. Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Lisboa, Portugal
Evaluation of nisin susceptibility of 219 Listeria spp. wild-type strains isolated from
milk, cheese and cheese processing environment at given conditions was the aim of
this work. Minimum inhibitory concentrations (MIC) were evaluated by the agar
incorporation method, on TSYEGA medium. The variability of Listeria spp. susceptibility
was very low especially at pH 5.5, with 98 % of isolates showing MIC values
between 10 and 50 IU ml -1 at 37 °C. The increase of pH from 5.5 (average of cheese
core pH) to 6.8 (average of cheese rind pH) at 37 °C resulted in an increase in the
MIC values by 5 to 20 times (from 10 – 50 to 50 – 100 IU) or more (> 100 IU ml 1) in
68 % of isolates. An increase in temperature from 20 °C to 37 °C at pH 5.5 resulted
in an increase on MIC values only in 8 % of all isolates tested. Nisin spontaneous resistance
at a frequency of 10 -3 to 10 -4 was observed more frequently at pH 6.8 and
37 °C. Exposure to combined stress conditions different from the ones tested
herein was previously reported to increase cell resistance mechanisms and indicates
that changes in virulence may occur concomitantly. Nevertheless, the majority
of strains tested were able to grow at 10 IU nisin ml -1 but not at 50 IU ml -1 at pH
5.5, showing a very susceptible profile. The lower pH (5.5) and temperature (20 °C)
tested, offered better conditions to the inhibitory action of nisin which demonstrated
a good potential for use in bioactive coatings as a combined hurdle effect to
control L. monocytogenes strains in dairy products.
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R E F E R E N C E
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AREA // E // Communication, risk perception and consumer practices – Social sciences in Listeria control
POSTER PRESENTATIONS // P / 178–181
What is an appropriate level of protection for Listeria monocytogenes
in foodstuffs consumed by vulnerable groups?
Little, C., Gillespie, I., Grant, K., Gormley, F., Mook, P. and McLauchlin, J.
Health Protection Agency Centre for Infections, UK
It is widely recognised that the consumption of contaminated food is an important
route of transmission of listeriosis and a wide range of food products have
been shown to be associated with both outbreaks and sporadic cases. Food safety
criteria for L. monocytogenes in Regulation (EC) No. 2073/2005 are applicable to
three categories of ready-to-eat (RTE) foods: absence of L. monocytogenes in 25 g is
required in RTE foods intended for infants and those for special medical purposes;
while for all other RTE foods L. monocytogenes should not exceed 100 cfu/g within
shelf-life. However, vulnerable groups (pregnant women, the immunosuppressed,
the elderly, and many patients in hospitals) are at particular risk of infection, and
consumption of low levels of L. monocytogenes may be of greater risk when eaten
by these groups. Source attribution of L. monocytogenes infections in England and
Wales to RTE food sources has shown that the most important source for the overall
population were multi-component foods (e.g. sandwiches, pre-packed mixed
salads) (23.1 %). Attribution of major sources was similar for the elderly population
(multi-component foods (22.0 %)). However, for pregnant women, beef (12.3 %),
milk/milk products (11.8 %) and fish (11.2 %) were more important sources of infection.
Concurrently, a number of outbreak investigations in England, Wales and
Northern Ireland have revealed that infections were linked to consumption of
sandwiches served to the vulnerable group population in hospital settings. Sandwiches
tested in these investigations contained the outbreak strain of L. monocytogenes
at very low concentrations (present at < 10/g) and legally would be deemed
safe by the Regulation. However, the legal limit of 100 cfu/g is not based upon formal
dose-response formulas and given the recognised rise in listeriosis observed in
the EU, it is questionable whether the legal limit affords the level of protection
deemed appropriate to protect the vulnerable group population.
ILCD: An Interactive Listeria culture diversity knowledgebase
Ashok Kumar, J. 1, Barbuddhe, S. B. 1, Kalekar, S. 1, Rodrigues, J. 1, Chopade, N. A. 2, Hain, T. 3
and Chakraborty, T. 3
1. ICAR Research Complex for Goa, Ela, Old Goa
2. Department of Pathology, Nagpur Veterinary College, Maharashtra Animal and Fishery Sciences
University, Nagpur, India
3. Institute of Medical Microbiology, Justus Liebig University, Giessen, Germany
Indian Listeria Culture Database (ILCD) is an online databank of profiles developed
for the Listeria strains isolated in India from various sources. ILCD is based
on a relational database management system that is hyperlinked to visualize phenotyping
and genotyping results in the form of tables and DNA fingerprint images
for individual strains. The system has been developed using open source LAMP
(Linux, Apache, MySql and PHP) tools. The database contains geographical source
of the strain, its lineage, serotype, source of isolation (animal/human), year of isolation,
phenotypic and genotypic characteristics, antibiotic sensitivity patterns,
and DNA fingerprint (PFGE image). This is an interactive web based database so
that the data can be exchanged between laboratories electronically. A flexible
search system based on systematic comparisons of the database entries allows
inter-laboratory comparison of profiles. There are search options, state wise,
serotype wise and so on. The parameters can be selected to search the information
required. Whole description of a particular strain can be visualized by clicking on
identity number of the strain. The data can be stored and accessed via internet
browser. The web interface of ILCD allows users with no special programming
background or bioinformatics experience to examine the results. The user is able
to view the description of any particular strain e.g. isolated from human or animal
or food source; or retrieve all information about a particular strain. Being the
first of its kind, ILCD is expected to be a very helpful tool in strengthening the
concept of ‘geographic genomics’ and will be very helpful to molecular epidemiologists
and those interested in research on Listeria.

AREA // E // Communication, risk perception and consumer practices – Social sciences in Listeria control
POSTER PRESENTATIONS // P / 178–181
Listeria spp. and the domestic environment: consumer knowledge,
attitudes, risk perceptions and food-handling behaviours
Redmond, E. C.
Cardiff School of Health Sciences, UK
In Europe, since 2000 incidence of listeriosis has increased by >59 %. This increase
has occurred almost exclusively in adults aged > 60 years with listerial bacteremia.
The importance of the home as location for acquiring foodborne disease
has prompted numerous studies to understanding of consumer food safety practices
in the domestic environment. Cognitive and behavioural data is required to
develop effective communication initiatives to improve food-handling practices
and decrease incidence. This study aims to review microbiological studies and determine
consumer risk perceptions, knowledge and domestic food-handling behaviours
of that may contribute to the risk of listeriosis. Electronic searches of
the Internet and library databases, personal-communication with food safety professionals
and attendance at international conferences has facilitated the collection
of 143 cognitive and behavioural consumer food safety studies and 16 microbiological
surveys of the domestic environment carried out over the past 35years.
Listeria has been isolated from up to 62 % domestic kitchens and frequently contaminated
items include refrigerators (2-10 %isolations) and kitchen dishcloths/sinks
(10-84 %isolations). Cumulatively, 8 % of all consumer food safety
studies investigated food safety of older adults; 4 % of pregnant women and 16 %
other consumer groups. Data indicates fewer (17 %) older adults are aware of Listeria
compared to other consumer groups (32-87 %). Similarly, 83 % adults (aged >
60years) lack knowledge of correct refrigerator temperatures, with > 80 % temperatures
exceeding recommendations. Observed risk-related handling malpractices
implemented by adults aged 60-75years are linked to pathogenic contamination
of kitchen surfaces and ready-to-eat (RTE) foods. Considerable
inadequacies in food safety practices and behavioural influences have been identified
that may increase risk of listeriosis. Data comparisons between consumer
groups will be presented, including likelihood of consuming RTE foods past useby-dates,
frequency of checking refrigerator temperatures and cleaning of refrigerators.
Findings will be discussed in the context of strategy development and future
consumer food safety communication iniatitives designed to decrease
incidence of listeriosis.
Do you know the temperature in your refrigerator?
Røssvoll, E. 1, Jacobsen, E. 2, Ueland, Ø. 1, Einar Granum, P. 3 and Langsrud, S. 1
1. Norwegian Institute of Food, Fisheries and Aquaculture Research
2. SIFO, National Institute for Consumer Research, Norway
3. Norwegian School of Veterinary Science
The use of legislation to reduce risk is at present used at all steps of the food production
chain from the primary producer to the stores. It is, however, both difficult
and undesirable to manage consumer food handling through legislation. As soon as
the products are bought by the consumer, the knowledge about how the product is
treated further terminates. The aim of this study was to improve food safety in
the domestic environment by risk analysis of consumer food handling and evaluation
of risk-reducing measures. We wanted to get a better understanding of the
interaction between consumer food handling practices and the potential growth of
pathogens under domestic conditions. To investigate this we conducted a consumer
survey, a case study, and a microbial experiment and showed that 1) consumers
were ignorant to or unaware of the temperature in their refrigerator, 2)
ready-to-eat (RTE) foods were exposed to great temperature variations by consumers,
and 3) the fluctuations in temperature resulted in enhanced growth of
psychrophilic pathogens as Listeria monocytogenes. In conclusion, consumers expose
themselves to increased risk of contracting food borne diseases by being ignorant
to or unaware of the temperature in their refrigerators. The temperature
fluctuations found in the case study enhanced pathogenic growth, and can in worst
cases lead to outbreaks of food borne diseases.
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R E F E R E N C E
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R E F E R E N C E
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47
AREA // A // Biology of Listeria monocytogenes
POSTER PRESENTATION// A/ 47
Listeria monocytogenes agr system: Quorum sensing, or maybe not?
Garmyn, D., Révelin, C. and Piveteau, P.
Université de Bourgogne – INRA, France
Communication, often referred as Quorum Sensing, is involved in the adaptation
of most bacteria to their environment. So far, the agr system is the sole communication
system described in the genus Listeria. The agr communication system affects
the biology of Listeria monocytogenes during saprophytic life (biofilm formation)
and during infection. We have recently demonstrated that agr expression
was heterogeneous during growth of L. monocytogenes EGDe as biofilm. Our observations
did not fit the criteria of the Quorum Sensing paradigm. Indeed, Quorum
sensing means that the whole population presents the same phenotype once
the quorum is reached. In order to investigate whether the agr system fits the Quorum
Sensing paradigm, or not, we investigated agr expression in situ by combining
gfp reporters, flow cytometry and fluorescent microscopy. This experimental set
up was used to revisit auto-induction, Quorum Sensing and more recent theories
of bacterial communication during the saprophytic life of Listeria monocytogenes.

AREA // D // Strategies for prevention and control of Listeria monocytogenes
POSTER PRESENTATIONS // P / 199– 200
Utilization of Lactococcus lactis M104, a wild nisin-producing raw milk
isolate, as an antilisterial adjunct in traditional Greek Graviera cheese
processing
Samelis, I., Pappa, E., Bogovic-Matijasic, B. and Rogelj, I.
National Agricultural Research Foundation, Dairy Research Institute, Greece
Recent validation studies have shown that growth inhibition of listerial contamination
is assured during processing and storage of traditional Greek Graviera
cheese in compliance with the current E.U. regulatory criteria for a maximum Listeria
monocytogenes population of 100 cfu/g allowable in ready-to-eat foods. Since,
however, the pathogen survived well during ripening, this study aimed at enhancing
L. monocytogenes inactivation by using Lactococcus lactis M104, a nisin-producing
(Nis+) raw milk isolate, as an antilisterial adjunct at cheese manufacture. Fresh
Graviera curds with added a commercial starter culture (SC), or the SC plus the
Nis+ strain (SC+M104), were inoculated (3 log cfu/g) with a 3-strain cocktail of
avirulent L. monocytogenes/innocua before molding in a commercial plant. After
brining, cheeses were ripened at 17-18ºC and 90% RH for 20 days and stored at 4ºC
in vacuum. The fate of Listeria was monitored during cheese ripening (0, 1, 3, 5, 11,
17, and 23 days) and storage (40 and 60 days). Changes in lactic acid bacteria (LAB)
populations and main chemical parameters were also monitored, while the Nis+
gene was detected in ripened cheeses and their LAB consortia by PCR. Populations
of Listeria indicators did not grow in the cheeses at any stage; they declined 10-fold
(P < 0.05) within the first 5 days and survived with little death thereafter. Although
Nis+ colonies and the Nis+ gene were isolated from the SC+M104 cheeses only, no
major differences in Listeria survival occurred between cheeses with or without
the Nis+ strain. All cheeses contained enterocin (A, B and P) plus plantaricin A
genes associated with Enterococcus faecium and Lactobacillus plantarum of the
NSLAB flora, and showed similar chemical changes. Thus, conditions prevailing
during Graviera cheese processing might suppress nisin (Nis+) expression by L.
lactis M104 at levels insufficient to deliver or enhance Listeria inactivation.
The European Project BASELINE “Selection and improving of fit-forpurpose
sampling procedures for specific foods and risks”
Manfreda, G. and De Cesare, A.
Department of Food Science – Alma Mater Studiorum University of Bologna, Italy
Food Safety Objectives (FSOs) and Performance Objectives (POs) are new criteria
complementing the existing concepts of microbiological criteria. To achieve these
objectives it is critically important a harmonisation of food safety control procedures.
The EU project “Selection and improving of fit for purpose sampling procedures
for specific foods and risks”-BASELINE, funded under the FP7 programme,
intends to obtain the following objectives: 1) to review the sampling
schemes currently available for food authorities and food producers to collect data
for quantitative risk assessment at EU level; 2) to assess the relevance and suitable
limit values of POs and FSOs for biological risks, including Listeria monocytogenes;
3) to evaluate the need for new or adapted methods for sampling and testing the
identified biological risks; 4) to develop predictive mathematical models for the
target biological risks; 5) to validate and harmonise the sampling schemes and the
alternative detection methods developed in the project; 6) to share and disseminate
the scientific knowledge coming out from the project to stakeholders. The
project results will be translated in clear recommendation to the EC as well as end
users and they will have a significant impact on protection of human health. A
poster detailing the project objectives, strategies and expected impact will be presented
at the conference.
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AUTHORS INDEX

Abdalla, S. D/P/128
Abee, T. A/O/05
Acciari, V. C/P/184, D/P/196
Achtman, M. A/P/27
Agyekum, K. B/O/14
Aharonowitz, Y A/O/11
Aish, J. C/P/125
Albano, H. D/P/163
Alessandria, V A/P/32, A/P/38, B/P/64, D/P/164
Allain, V. D/P/190
Allen, V. A/P/29
Almeida, G. A/P/30, A/P/44, C/P/105, C/P/111,
C/P/113, D/P/166, D/P/165
Almeida, M. T. B/P/66
Alonzo, F. B/P/56
Alvarez-Dominguez, C. A/P/00, B/O/19, C/PL/09,
Amagliani, G. C/P/93
Amar, C. C/P/91, C/P/92
Amar, C. F. C/P/85
Ambrosoli, R. D/P/150
Ancora, M. C/P/102
Andersen, J. B. B/P/182
Andrew, P. B/P/54
Andrew, P. D/P/128
Andrew, P. W. A/P/10
Anita, B. A/P/12, A/P/19
Arapakh, S. D/P/172
Armillotta, G. C/P/185
Arnau, J. D/P/136
Arneborg, N. D/P/132
Asakura, H. A/P/22
Ašanin, R. D/P/177
Ashok Kumar, J. C/O/25, E/P/179
Asséré, A. D/P/146
Atanassova, V. D/P/173
Aubry, C. B/O/21
Auchter, M. B/P/50, D/P/148
Aury, K. D/P/190
Aymerich, T. D/O/43, D/P/136
Azeredo, J. C/P/115, D/P/176
Azevedo, I. A/P/30
Bai, F. A/P/36
Barbosa, J. A/P/30, C/P/111, C/P/113, D/P/160,
D/P/163, D/P/165
Barbuddhe, S- B. A/P/06, A/P/07, B/P/49, C/O/25,
C/P/106, C/P/107, C/P/108, C/P/117, D/P/157, E/P/179.
Barile, M. D/P/129
Barre, L. C/P/103
Batista, S. A/O/02
Baysal, A.H. C/P/114
Beaufort, A. D/O/40, D/P/154
Belessi, C. A. D/P/172
Belletti, N. D/O/43
Benjamin, F. D/P/146
Berg, J. D/P/141
Bergis, H. D/O/40, D/P/154
Bergmann, S. B/P/76
Bergstrøm, A. B/P/182
Bernardi, T. D/P/145
Berrang, M. E. D/P/126
Berzins, A. D/P/155
Besse, N. G. C/P/103
Bhosle, S. N. C/P/107, C/P/108
Bianchi, D. M C/P/123
Bielecka, M.K. C/O/24
Bitar, A. P. B/P/58, B/P/59
Blatter, S. D/P/131
Block, C. S. C/O/29
Boelaert, F. C/O/32, D/P/170
Boone, R. C/P/94
Boor, K. J. A/O/13, A/P/31
Borges, S. A/P/30, D/P/163
Borza, A. C/P/119, C/P/120
Boscher, E. A/P/04
Bosley, J. C/P/119
Botello-Morte, L. A/P/43
Bottero, M. T. C/P/124
Boucheix, C. B/O/20
Bover-Cid, S. D/O/43
Bowman, J. P. A/O/03, A/P/17
Boye, M. B/P/182
Brandi, G C/P/93
Braun, E C/O/29
Bremont, S. C/P/90
Brennan, O. A/O/09
Briandet, R. D/P/142
Briers, Y. A/O/07
Brisabois, A. A/P/01, B/P/47, C/P/82, D/P/146
Brisse, S. A/O/06
Brito, L. A/O/02, D/P/153, D/P/167
Bruno, J. C. B/O/22
Bugarski, D. D/P/177
Burall, L. A/P/26
Burgess, S. C. B/P/48
Busia, G. D/P/175
Butler, F. D/O/42
Byrne, B. C/P/119
Cabanes, D. B/O/23, B/P/65, B/P/66, B/P/68, B/P/70
Cabanova, L. D/P/152
Cabo, M.L. A/P/45
Cabrita, P. A/O/02
Caccia, N. D/P/142
Calderone, D. D/P/194
Calendar, R. A/O/10
Calvo, E. A/P/43
Camejo, A. B/O/23, B/P/70
Cammá, C. C/P/102
Campos, L. D/P/189
Campos, M. L. D/P/188
Candeloro, L. C/P/186
Cantinelli, T. A/O/06
Carneiro, L. A/P/44, D/P/166
Caro, V. A/O/06
Carpentier, B. C/P/103
Carranza-Cereceda, C. A/P/00, B/O/19
Carrasco-Marin, E. A/P/00, B/O/19
Carrera, S. A/P/45
Cartinhour, S. A/O/13
Carvalho, F. B/O/23
Carvalho, F. B/P/65
Casey, P. G. B/P/69
Castle, M. E/O/49
Cécile, C. D/P/171
Ceres, C. D/P/129
Ceri, H. D/P/176
Chafsey, I. A/P/08, D/P/142
Chakraborty, T. A/PL/01, B/P/77, B/P/78,
C/O/25, C/P/106, E/P/179
Chambom, C. A/P/08
Chamot, S. D/P/145
Chaturongakul, S. A/P/31
Chaudhuri, S. B/P/71
Chavant, P. D/P/145
Chaves-Lopez, C. D/P/151
Chemaly, M. D/P/147
Chemaly, M. M. C/P/183, D/P/187, D/P/190
Chen, J. A/O/04, A/P/36
Chen, Y. A/P/16
Chenal-Francisque, V. A/O/06, C/P/100, D/O/46
Chiarini, E. D/P/134
Chopade, N. A. E/P/179
Christensen, B. B. B/P/182
Civera, T. C/P/123, C/P/124
Clark, A. D/O/38
Cocolin, L. A/P/32, A/P/38, D/P/150, D/P/164
Colaneri, C. A/P/01
Comaposada, J. D/P/136
Commeau, N. D/P/154
Corbett, D. B/P/63
Corde, Y. B/P/47
Cormica, M. G. C/P/110
Cornu, M. D/O/40, D/P/154
Correia, C. D/P/188, D/P/189, D/P/191
Cortesi, M. L. D/P/129
Cossart, P. B/O/20
Costas, B. D/O/37
Courvalin, P. A/P/01, C/P/90
Courtillon, C. C/P/183, D/P/187

Couture, H. C/P/97
Crerar, S. E/O/49
Cross, L. C/P/92
Cummins, J. B/P/62
Cunha, C. I. D/P/189, D/P/191
Czuprynski, C. B/P/60
D’Orazio, V. A/P/43
da Silva Felício, M.T C/O/32
da Silva, M. B/O/16
Dal Bello, B. D/P/150
Dalgaard, P. D/O44
Dalmasso, A. C/P/124
D’Amico, D. C/P/112
Daneshvar, M. I. A/P/33
Daniel, P. D/P/171
Danielsson-Tham, M.-L. C/O/30, C/P/122
Datta, A. A/P/26, B/P/67
David, C. B/P/54
Davis, A. D/P/159
D’Costa, D. C/P/106, C/P/108
De Cesare, A. C/P/116
de Courseulles, E. D/P/192
De Martinis, E. C. D/P/148
De Valk, H. C/O/31, C/P/100
Dear, P. C/P/85
Decastelli, L. C/P/123
DeLappe, N. C/P/110
Dell’Era, S. A/O/07
Den Bakker, H.C. A/P/33, D/P/144
Deng, X. A/O/08
Desai, M. C/P/92
Deschamps, J. D/P/142
Destro, M. T. D/P/134, D/P/135
Desvaux, M. A/P/08, D/P/142, D/P/145
Desvaux, M. D/P/142
Dhuri, R. B. C/P/108
di Febo, T. C/P/185
di Giannatale, E. C/P/186, D/P/196, D/P/197
di Pasquale, F. C/P/101, D/P/151
Disson, O. C/O/24
Doijad, S. C/P/107, C/P/108, D/P/157
Dolci, P. D/P/164
Donaldson, J. R. B/P/48
Donnelly, C. C/P/112
Dorey, M. C/P/119, C/P/120
Douey, D. C/P/119, C/P/120
Doux, C. D/P/192
Dowd, G. B/P/69
Duarte Cruz, C. B/P/53
Dumas, E. A/P/08
Dusch, V. D/O/46, C/P/100
Dussurget, O. B/P/55
Ebersbach, T. B/P/182
Egorova, I. C/P/95, C/P/96
Einar Granum, P. E/P/181
Eiriz, E. A/P/45
Eisenreich, W. B/O/18
Elemam M. M. C/P/88
Eleni, S. A/P/12
Eliav, H. C/O/29
Ells, T. C. A/P/40
Eloranta, K. C/P/120
Endres, J. D/P/148
Engeljohn, D. D/PL/11
Ermolaeva, S. A/P/34, B/O/15, B/P/73
Eugster, M. R. A/O/10, A/P/28
Eusébio, C. A/P/44
Eylert, E. B/O/18
Fablet, A. D/P/147
Faith, N. B/P/60
Faith, N. G. B/P/74
Faleiro, M. L. A/P/10
Fall, P. A. D/P/137
Fang, W. A/O/04, A/P/36
Farber, J. A/P/09, C/PL/08, C/P/97, D/P/143
Fávero, L.M. D/P/139
Favretti, M. C/P/84
Felício, T. D/P/170
Félix, B. C/P/82
Fernandes, L. P. D/P/140
Fernandez Guerrero, M. L. B/P/75
Fernandez-Prieto, L. A/P/00, B/O/19
Ferreira, I. D/P/188, D/P/189
Ferreira, M.A.S.S. A/P/42, D/P/198
Ferreira, P. B/O/23
Ferreira, R.B. A/O/02
Ferreira, V. A/P/30, D/P/160
Ferreira-Dias, S. D/P/153
Ferrini, G. D/P/136
Fertikov, V. C/P/96
Filiatrault, M. J. A/O/13
Fletcher, G. B/P/53
Flieger, A. A/P/03
Foglini, M. C/P/93
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Wiedmann, M. A/PL/02, A/O/13, A/P/16,
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Wilhelm, T. C/P/122
Williams, D. B/O/14
Winkelströter, L. K. D/P/148
Wong, H. T. L. A/P/39
Wren, B.W. A/P/05
Xayarath, B. B/P/57
Yurov, D. A/P/34
Zahle Andersen, A. A/P/24
Zaytseva, E.A. B/P/73
Zeiman, E. A/O/10
Zeppa, G. D/P/150
Zhang, W. A/O/08
Zuliani, V. D/P/154
Zurbriggen, A. B/P/52
Zweifel, C. D/P/131

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Universidade Católica Portuguesa – Escola Superior de Biotecnologia
Porto, 2010